Mechanisms underlying KCNQ1channel cell volume sensitivity Hammami.pdfThis thesis starts with a general introduction to ion channels followed by an overview of mechanosensitive ion

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T H E P H D S C H O O L O F S C I E N C E

F A C U L T Y O F S C I E N C E

U N I V E R S I T Y O F C O P E N H A G E N

PhD thesis

Sofia Hammami

Mechanisms underlying KCNQ1channel

cell volume sensitivity

Submitted: 10/05/10

TABLE OF CONTENTS Preface ............................................................................................................................................................... 5

Acknowledgements.......................................................................................................................................... 5

Publications ....................................................................................................................................................... 6

Summary ............................................................................................................................................................ 7

Dansk resumé ................................................................................................................................................... 8

Abbreviations ................................................................................................................................................... 9

Table of figures .............................................................................................................................................. 10

BACKGROUND .............................................................................................................................. 11

Ion Channels ................................................................................................................................................... 11

Mechanosensitive ion channels ................................................................................................................... 12

Cell volume sensitive ion channels .................................................................................................... 17

The KCNQ1 channel ........................................................................................................................... 18

1. Expression and role in epithelia and cardiac tissue ........................................................... 19

2. Regulation ................................................................................................................................ 20

a. Regulation by β subunits ................................................................................................... 21

b. Regulation by cell volume ................................................................................................. 23

Role of volume sensitive KCNQ1 in mammary epithelium ............................................ 23

Role of volume sensitive KCNQ1 in liver cells ................................................................. 24

Role of volume sensitive KCNQ1 in cardiomyocytes ...................................................... 24

Purinergic receptors and ATP signalling .................................................................................................... 25

Receptors .................................................................................................................................................... 25

ATP release mechanisms ......................................................................................................................... 26

THESIS OBJECTIVES .................................................................................................................... 28

METHODS ........................................................................................................................................ 29

Two-Electrode voltage clamp technique (TEVC) .................................................................................... 29

The patch clamp technique .......................................................................................................................... 30

ATP bioluminescent assay ............................................................................................................................ 30

Enzyme linked immunoassay for surface expression .............................................................................. 31

RESULTS AND DISCUSSION ..................................................................................................... 32

Cell swelling vs. membrane stretch ............................................................................................................. 33

ATP release and cell volume changes ......................................................................................................... 35

KCNQ1 association with KCNE1 and volume sensitivity ..................................................................... 36

The cytoskeleton ............................................................................................................................................ 38

Intracellular calcium ...................................................................................................................................... 38

Cytosolic pH ................................................................................................................................................... 38

Membrane PIP2 .............................................................................................................................................. 39

Kinases ............................................................................................................................................................. 39

Specific residues for the volume sensitive potassium channels .............................................................. 40

CONCLUSION ................................................................................................................................. 42

REFERENCE LIST ......................................................................................................................... 43

APPENDIX ....................................................................................................................................... 56

Manuscript I: Cell volume and membrane stretch independently control K +

channel activity .................................................................................................................... 57

Manuscript II: KCNQ1 channel response to cell volume changes is not mediated

by ATP release..................................................................................................................... 64

Manuscript III: KCNE1-induced increase in KCNQ1 currents is not mediated

through enhanced plasma membrane expression .......................................................... 86

Related paper: Cell swelling and membrane stretch – A common trigger of

potassium channel activation? ....................................................................................... 105

PhD thesis Sofia Hammami

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Preface

This presented PhD thesis is the result of three years of work under supervision of Associate

Prof. Niels J. Willumsen and Prof. Ivana Novak at the Department of Biology as well as

Prof. Dan A. Klærke from the Department of Physiology and Biochemistry, IBHV, at LIFE.

This thesis starts with a general introduction to ion channels followed by an overview of

mechanosensitive ion channels with emphasis on volume sensitive potassium channels.

Subsequently, I give a description of the KCNQ1 channel and how it is regulated by KCNE1

and by cell volume. This is followed by a brief description of purinergic receptors and ATP

signalling. The basics of the different techniques used are described in the method section.

Attached to the thesis, are one published article and two other submitted manuscripts. In the

discussion, the main findings from these studies are briefly discussed and other possible

mechanisms will also be included. In addition, I have attempted to propose different

perspectives related to the further identification of the so far unknown mechanisms behind

channel activation upon small, fast changes in cell volume.

Acknowledgements

Many people are deserving acknowledgement at this time for their help in making this

project possible. First and foremost, I would like to thank my supervisors, Niels and Dan,

for excellent guidance and continuous support. It has been a great pleasure to be under their

wings during my master and PhD for almost 5 years. I also owe a great thank to Prof. Ivana

Novak for the kind supervision and support I received from her during the last 1½ year of

my work.

I would like to thank all people from the 3rd floor at August Krogh building for a great

working environment as well all the members of Dan Klærkes lab, former as well new

members. A special thank goes to Zaida Rasmussen for technical help and support.

Last, but not least, I‟m thankful to my family, friends and especially Martin for giving me

moral support and for always being there when I needed him.


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Publications

My work as a PhD student has resulted in 1 published manuscript, 3 published abstracts and

2 submitted manuscripts. Additionally I was invited to write a short review article related to

my first manuscript to be published in Physiology News magazine.

1 published article, 2 manuscripts and the related review article are included in the thesis

(attached in Appendix):

I. Hammami S, Willumsen NJ, Olsen HL, Morera FJ, Latorre R, & Klaerke DA (2009). Cell volume and membrane stretch independently control K+ channel activity. J Physiol 587, 2225-2231.

II. Hammami S., Willumsen NJ, Klaerke DA, & Novak I. (2010). KCNQ1 channel

response to cell volume changes is not mediated by ATP release. (To be submitted)

III. Hammami S, Klaerke DA & Willumsen NJ (2010). KCNE1-induced increase in

KCNQ1 currents is not mediated through enhanced plasma membrane expression

(submitted)

Related paper: Cell swelling and membrane stretch – A common trigger of potassium channel activation?


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Summary

Cells are constantly exposed to changes in cell volume during cell metabolism, nutrient uptake, cell proliferation, cell migration and salt and water transport. In order to cope with these perturbations, potassium channels in line with chloride channels have been shown to be likely contributors to the process of cell volume adjustments. A great diversity of potassium channels being members of either the 6TM, 4 TM or 2 TM K+ channel gene family have been shown to be strictly regulated by small, fast changes in cell volume. However, the precise mechanism underlying the K+ channel sensitivity to cell volume alterations is not yet fully understood.

The KCNQ1 channel belonging to the voltage gated KCNQ family is considered a precise sensor of volume changes. The goal of this thesis was to elucidate the mechanism that induces cell volume sensitivity. Until now, a number of investigators have implicitly assumed that changes in cell volume are associated with parallel changes in membrane stretch, and, consequently, that regulation by cell volume and by membrane stretch constitute a common regulatory mechanism. This assumption was challenged in Manuscript I where we analyzed and compared the effects of (1) osmotic cell swelling and (2) local membrane stretch on the highly volume sensitive KCNQ1 channel and the highly stretch sensitive BK channel. In this study we present evidence against this assumption by showing that activation of BK channels by local membrane stretch is not mimicked by cell swelling, and activation of KCNQ1 channels by cell volume increase is not mimicked by stretch of the cell membrane. Thus, we conclude that stretch- and volume-sensitivity can be considered two independent regulatory mechanisms.

Alternatively, volume-activation of ion channels could be mediated by an autocrine mechanism in which ATP released from the cells in response to volume changes activates signaling pathways that subsequently lead to ion channel stimulation. Whether volume sensitivity of KCNQ1 is modulated by ATP release was investigated in Manuscript II. ATP release from KCNQ1 injected oocytes was monitored by a Luciferin/Luciferase assay during cell volume changes and the effect of exogenously added ATP and apyrase on the cell volume induced KCNQ1 current was studied. Based on our data to date, we postulate that KCNQ1 does not seem to be responsive to ATP during cell volume changes, which indicates another mechanism of regulation.

Besides being regulated by cell volume, KCNQ1 is also modulated by the interaction of the β subunit KCNE1 giving rise to the cardiac IKs delayed rectifier potassium current. Apart from altering the kinetic characteristics of the KCNQ1 channel current, KCNE1 also augments the KCNQ1 current. It is debated whether this increase in macroscopic current upon expression of KCNQ1 with KCNE1 is due to an increase in ion channel conductance (γ), the open state probability (Po) or an increase in the number of channels in the plasma membrane (N). The latter was quantified by measuring the level of KCNQ1 surface expression by using an enzyme-linked immunoassay (Manuscript III). To do this, a HA-tagged version of the KCNQ1 channel was expressed with and without KCNE1 in Xenopus oocytes. The results show that the KCNQ1 surface expression was significantly lower when KCNE1 is coexpressed compared to KCNQ1 alone despite the higher current for the heteromeric KCNQ1/KCNE1. This indicates that the overall increase of the KCNQ1 current, when KCNE1 is coexpressed, is not due to an increase in ion channel surface density but rather to an increase in single-channel conductance or in open state probability.


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Dansk resumé

Celler er konstant udsatte for volume ændringer ved celle migration, metabolisme, proliferation og ved optagelse af næringsstoffer samt salt og vand transport. For at klare disse forstyrrelser, har kalium kanaler, på samme vis som kloridkanaler, vist sig at være væsentlige i tilbage justering af celle-volumen og dermed bidrage til den regulatorisk volume proces. En stor del af kalium kanaler, som er medlem af enten 6TM, 4 TM eller 2 TM gen familierne har vist sig at være nøje reguleret af små, hurtige ændringer i cellevolumen. Imidlertid er den mekanisme der ligger til grund for kalium kanalers følsomhed over for celle-volume ændringer, endnu ikke helt forstået.

KCNQ1 kanalen, der tilhører den spændingsafhængig KCNQ familie betragtes, som en præcis sensor af volume-ændringer. Målet med denne afhandling er at klarlægge den mekanisme, der inducerer kanalens celle-volume følsomhed. Indtil nu har mange studier implicit antaget, at ændringer i cellevolumen er forbundet med parallelle ændringer i membran stræk, og derfor, at de begge reelt udgør en fælles reguleringsmekanisme. Denne antagelse bliver udfordret i manuskript I, gennem en analyse og sammenligning af virkningerne af (1) osmotisk celle svulmning og (2) lokalt membran stræk på den volumen følsomme KCNQ1 kanal samt den stræk-følsomme BK kanal. I denne undersøgelse præsenteres beviser mod denne antagelse. Der vises, at aktivering af BK kanaler ved membran stræk ikke er efterlignet af membranspænding fremkaldt af celle svulmning, samt at aktivering af KCNQ1 kanaler ved celle svulmning ikke er medieret af de lokale spændinger i celle membranen. Således konkluderes, at stræk og volumen-følsomheden skal betragtes som to uafhængigt af hinanden regulerende mekanismer.

Alternativt, kunne aktivering af ion kanaler ved volumen ændringer, være medieret af en autocrine mekanisme, hvor ATP frigives fra cellerne og aktiverer signalveje, som derefter fører til stimulering af ion kanaler. Hvorvidt den volumen-følsomme KCNQ1 moduleres af ATP frigivelse undersøges i Manuskript II. Ved denne undersøgelse blev ATP frigivelse fra oocyter, der udtrykker KCNQ1, målt ved hjælp af Luciferin/luciferase assay ved forskellige volume ændringer. Endvidere, blev virkningen af eksogent tilsat ATP og apyrase undersøgt på KCNQ1 kanal strømmen. Baseret på disse data, er det vores opfattelse, at KCNQ1 ikke er reguleret af ATP under ændringer i cellevolumen.

Ud over at være reguleret af celle volume ændringer, er KCNQ1 også moduleret af β- subunit KCNE1. Når disse udtrykkes sammen, giver det ændringer i de kinetiske egenskaber af KCNQ1 kanalen herunder en væsentlig forøgelse af KCNQ1 strømmen. En del studier har diskuteret om denne stigning i makroskopisk strøm skyldes en stigning i ion kanal konduktansen (γ), åbnings sandsynligheden (Po) eller en stigning i antallet af kanaler i plasmamembranen (N). Sidstnævnte blev i dette studie kvantificeret ved, at måle niveauet af udtrykte KCNQ1 kanaler på membranoverfladen ved hjælp af enzym-linked immunoassay (Manuskript III). For at gøre dette, er en HA-mærket KCNQ1 kanal blev udtrykt med og uden KCNE1 i Xenopus oocyter. Resultaterne viser, at KCNQ1 overflade ekspressionen blev markant lavere, når KCNE1 er co-udtrykt sammenlignet med KCNQ1 alene. Dette på trods af, at der måles en højere strøm når KCNQ1 og KCNE1 er udtrykt sammen. Dette viser, at den samlede stigning i KCNQ1 strømmen, ved co-udtrykt KCNE1 ikke skyldes en stigning i ion kanalens overflade ekspression, men snarere skyldes en stigning i enkelt-kanal konduktansen eller i åbnings-sandsynligheden.


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Abbreviations

AA Amino Acids AC Adenylate cyclase AMP Adenosine monophosphate ADP Adenosine Diphosphate AP Action Potential AQP Aquaporin ATP Adenosine Tri-Phosphate BK Big Conductance calcium-

activated Potassium channel BNFC Benign Neonatal Familial

Convulsions cAMP Cyclic AMP CFTR Cystic Fibrosis

Transmembrane Regulator CHIF Corticosteroid hormone

induced factor ClC Chloride channel DAG Diacylglycerol DFNA Deafness autosomal

dominant nonsyndromic sensorineural

ENaC Endothelial Sodium channel HCN Hyperpolarization-activated

nucleotide-gated channel HERG Human Ether-a-go-go

related Gene IK Intermediate conductance

calcium-activated Potassium channel

IP3 Inositol Triphosphate JLNS Jervall-Lange-Nielsen Syndrome KCa

2+ Calcium activated potassium

channel KCNE K+ channel Nomenclature

family E, a family of β-subunits

KCNQ K+ channel Nomenclature family Q

Kir Inward Rectifier Potassium channel

Kv Voltage activated potassium LQT Long QT MAPK Mitogen Activated Protein

Kinase mOsm milli osmolarity MS Mechanosensitive NTPDase nucleoside triphosphate

diphosphohydrolases Po Open state probability PIP2 Phophatidylinositol 4,5

biphosphate PKA protein kinase A PKC protein kinase C PLC Phospholipase C RVD Regulatory Volume Decrease RVI Regulatory Volume Increase RW Romano-Ward SAC Stretch Activated Channel SK Small conductance Potassium TASK Twik-related Acid-Sensitive

K+ channel TEVC two electrodes Voltage Clamp TM Transmembrane TRAAK Twik related arachidonic

acid K+ channel TREK Twik related K+ channel TRP Transient receptor potential

channel TRPP TRP polycystin TRPV TRP vanilloid TWIK Tandem, weak inward

rectifier K+ channel UDP Uridine Diphosphate UTP Uridine-5'-triphosphate VRAC Volume regulated anion channel VSOR Volume sensitive outwardly

rectifying anion channel


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Table of figures

Figure 1 Studying mechanosensitivity of ion channels. ................................................................................ 14

Table 1 Overview of mechanosensitive ion channels. .................................................................................. 16

Figure 2 Overview of volume sensitive potassium channels across the K+ family tree. ......................... 18

Figure 3 Topology and channel architecture of KCNE and KCNQ1 proteins. ...................................... 19

Figure 4 Regulation of KCNQ1 by KCNE1 and cell volume. ................................................................... 21

Figure 5 Overview of purinergic receptors. .................................................................................................... 26

Figure 6 Measurement of surface expressed proteins through enzyme immunoassay. ........................... 31

Figure 7 Possible mechanisms for KCNQ1 cell volume sensitivity ........................................................... 41


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BACKGROUND

Ion Channels

All living cells are delimited by a plasma membrane separating the intracellular content from

their extracellular surrounding. The plasma membrane is composed of amphipathic

phospholipids where small uncharged molecules such as O2 and CO2 can easily cross the

membrane whereas other such as amino acids, ions, nucleic acids and carbohydrates, can

pass only with the aid of membrane proteins. Among these proteins are ion channels which

are integral membrane proteins forming pores that allow passage of inorganic ions mainly

Na+, K+, Cl- and Ca2+ across the membrane down their electrochemical gradients.

Ion channels are of pivotal physiological importance for many cellular functions such as the

regulation of the membrane potential, control of cardiac excitability, hormone secretion, cell

volume regulation, cell proliferation, intracellular signalling and many other biological

processes. Because of their great relevance and their specific expression in various tissue cells

and organs, ion channels are also involved in many pathophysiological conditions. Diseases

involving ion channel dysfunction due to mutations in the genes encoding for ion channels

are termed „Channelopathies‟ (Ashcroft, 2000). Today a multitude of human disorders

including epilepsy, cystic fibrosis, arrhythmias and many others have been linked to ion

channel dysfunction. Therefore ion channels have become major targets for a number of

therapeutic agents developed for the treatment of various diseases.

Three major methodological advances have been gathered around the study of ion channel

structure and function and have facilitated ion channel research during the past 35-40 years.

First the patch clamp technique invented by Bert Sakmann and Erwin Neher in 1976 allowed

ionic currents flowing through single channel proteins to be measured with unique precision

thereby deducting the single-channel conductance and channel kinetics. The technique is

based on making a high resistance seal, a so called giga seal between the glass electrode tip

and the cell plasma membrane by applying a negative pressure in the pipette. This high

resistance seal enables us to record small amplitudes of single channels in picoAmpere range

with very low level of background current noise. Second, advances in molecular genetics

have made it possible to clone individual channels and thus relate ion channel function to the

protein sequence from which they are constructed. Finally, another major breakthrough


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came in 1998 when Roderick MacKinnon‟s group used X-ray crystallography to resolve the

first three dimensional structure of an ion channel - the bacterial KcsA potassium-channel

from Streptomyces lividans (Doyle et al., 1998). This discovery provided an excellent model of

eukaryotic K+ channel architecture, an insight into how the ion specificity of channel

proteins is achieved and how the voltage sensor of voltage gated ion channels functions.

Three main features distinguish ion channels from other membrane proteins:

1. They conduct ions rapidly nearly as quickly as the ions move through free fluid;

2. They show ion selectivity permitting some ions to pass but not others. This depends

on the diameter and shape of the ion channel and on the distribution of charged

amino acid (AA) in its pore lining

3. They are gated i.e. they fluctuate randomly between two or more functional states,

usually an open and closed state by a change in confirmation. The transition between

the states is governed by the rate constants affecting the time spent by the channel in

the open or closed state. This open state probability is regulated by an external

stimulus such as ligand binding, membrane voltage, temperature and mechanical

stimulus such as stretch, shear stress or volume change depending on the type of

channel.

This thesis is focused on ion channels regulated by mechanical stimuli more precisely ion

channels sensitive to cell volume changes. A brief general introduction to mechanosensitive

ion channels is presented in the next section with focus on volume sensitive potassium

channels.

Mechanosensitive ion channels

Cells are constantly subjected to mechanical stimuli, such as changes in cell volume, stretch

and shear stress. For instance, changes in cell volume take place during physiological

processes such as secretion and salt and water transport in the intestine, kidneys and exocrine

glands, but may also occur during pathological conditions as in brain and heart ischemia,

diabetes and dehydration. Rhythmical stretch and relaxation of the lung epithelium occur

during breathing. However, this can be exaggerated during asthma and lung diseases. Shear

stress is likely to be seen in all kinds of tubular structures, such as kidney tubules as well as in

blood vessels due to the pulsatile nature of blood pressure and flow.


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Studies have revealed the presence of many intracellular and membrane bound components

that perceive and react upon such mechanical perturbations (Kalapesi et al., 2005). Today, a

great knowledge has been gathered about mechanosensitive (MS) ion channels and their

association to several major human diseases (Ingber, 2003), such as neuronal and muscular

degeneration, cardiac arrhythmias (Kohl et al., 2006), hypertension, polycystic kidney

diseases, atherosclerosis (Gautam et al., 2006), muscular dystrophy (Hamill, 2006), brain and

cardiac ischemia and much more (Ingber, 2003).

MS ion channels are of various ionic selectivities (Table 1), existing in more than 30 types of

cells, from animals to plants to fungi and even bacteria (Hu & Sachs, 1997;Sachs,

1988;Morris, 1990). One common characteristic for MS ion channels is that their gating is

altered in response to mechanical stimuli (membrane stretch, cell volume changes or shear

stress) generating an ionic current that is subsequently transformed into an electrical

response.

Early after the development of the patch clamp technique, the first recordings of cell

swelling and stretch-activated channel currents were obtained (Guharay & Sachs,

1984;Hamill, 1983). With the patch clamp technique the mechano-sensitivity of ion channels

can be studied while applying or subjecting the cells to different mechanical stimuli (see

figure 1).

The most studied class of MS channels is the stretch-activated channels (SAC) (Sachs &

Morris, 1998)(Table 1). SACs were first detected in chick skeletal muscles (Guharay &

Sachs, 1984). Their open state probability increases with increasing pressure applied at the

patch pipette. This mechanical stretch affect channel gating but without significant alteration

in current amplitude or conductance.


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Figure 1 Studying mechanosensitivity of ion channels.

Mechanosensitivity of ion channels can be studied by 1) exposing the cell to different extracellular

osmolarities thus provoking cell swelling or shrinkage; 2) Applying local pressure (stretch) in

membrane patches which are in contact with the pipette tip; and 3) inducing shear stress by exposing

the cell to fluid flow via the perfusion system (courtesy of N. Willumsen)

The mechanisms linking mechanical stimuli to the subsequent modulation of ion channels

are still not clearly understood and many possibilities are under discussion. Multiple studies

have provided evidence for the involvement of the underlying cytoskeleton, which exerts

forces on the channel leading to channel gating (Kalapesi et al., 2005;Jorgensen et al.,

2003;Grunnet et al., 2003;Grunnet et al., 2002b). Others postulate that solely membrane

tension mediates channel activation: reconstitution of completely functional stretch activated

bacterial channel into liposomes shows that membrane tension can directly be transferred to

the channel via the lipid bilayer independently of a underlying cytoskeletal network (Sukharev

et al., 1994;Sukharev et al., 1993;Markin & Martinac, 1991). Activation of membrane-bound

phospholipases during mechanical deformation was suggested to release fatty acids from the

membrane, which subsequently modulates ion channels (Kirber et al., 1992;Ordway et al.,

1995). Channel activation may also be a result of mechanical-induced stimulation of a

response that has no physical connection with the channel, for example Ca2+ release, ATP

release, phosphorylation or alteration of other signalling molecules (e.g. mitogen activated

protein kinase (MAPK), Protein kinase A and C) (Aikawa et al., 2002;Chen et al.,

1999;Giancotti & Ruoslahti, 1999). Other studies have revealed that shear stress provokes

bending of primary cilia which are non-motile structures projecting from the centriole e.g. in

Cell volume changes Suction Shear stress

Δ Osmolarity Negative pressure Fluid flow


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renal tubular cells, resulting in Ca2+ influx through mechanosensitive channels residing in the

cilium (Schwartz et al., 1997;Praetorius & Spring, 2001).

Table 1 on the next page lists some examples of ion channels that are either regulated by cell

volume changes, stretch or shear stress. This table does not include all known

mechanosensitive channels though the most important ones are mentioned.


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Table 1 Overview of mechanosensitive ion channels.

Type of ion channels Ion channel References

shear stress sensitive

Na+ channels

ENaC (Satlin et al., 2001)

K+ channels Kir2.1 IK

(Olesen et al., 1988;Hoger et al., 2002) (Brakemeier et al., 2003)

Cl- channels

Endothelial chloride channel Outward rectifying CLC

(Gautam et al., 2006)

Cation channels TRPV2 and TRPV4 , TRPpolycystin 1 and 2

(O'Neil & Heller, 2005)

Stretch sensitive

K+ channels (BK) (Kirber et al., 1992;Gasull et al., 2003)

Cl- channels CLC5 (Wang et al., 2010)

Cation channels Stretch activated cation channels (SACs)

(Hu & Sachs, 1997;Guharay & Sachs, 1984)

Volume sensitive

K+ channels SK and IK KCNQ1, KCNQ4, and KCNQ5 TASK-2, TREK-1 and TRAAK Slick (Slo2.1) Kir4.1 and Kir4.1-Kir5.1 Kv1.3 and Kv1.5

(Grunnet et al., 2002b;Jorgensen et al., 2003); (Grunnet et al., 2003;Jensen et al., 2005;Hougaard et al., 2004) (Kalapesi et al., 2005;Maingret et al., 2002;Lesage et al., 2000;Kelly et al., 2006;Niemeyer et al., 2001) (Personal communication-Stolpe K. and Tejada. M) (Soe et al., 2009) (Deutsch & Chen, 1993;Felipe et al., 1993)

Cl- channels Volume-regulated anion channels (VRAC) or volume sensitive outwardly rectifying anion channels (VSOR) Calcium activated chloride channel TMEM16

(Christensen & Hoffmann, 1992;Hoffmann & Pedersen, 2006;Pasantes-Morales et al., 2006) (Almaca et al., 2009)

Cation channels Hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2)

(Calloe et al., 2005)


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Cell volume sensitive ion channels

During physiological processes such as secretion, cell migration, growth, proliferation, cell

metabolism and salt and water transport, animal cells are constantly exposed to variations in

intracellular or extracellular osmolorarities resulting in smaller or larger changes in cell

volume. In the absence of any kind of mechanism for volume regulation, cells would swell

up to the point of lysis or shrink and loose normal functionality, therefore the maintenance

of a constant volume is very crucial for cell survival and proper cell function.

In order to counteract the changes in cell volume, cells respond to swelling and shrinkage by

processes called regulatory volume decrease (RVD) and increase (RVI), respectively. During

cell volume increase, potassium and negatively charged ions (KCl) exit the cell, thereby

decreasing the osmolarity of the cytosol. Subsequently, water flux out of the cell to drive

volume recovery. Osmotically shrunken cells, in contrast, initiate a gain of KCl and water

thereby increasing cell volume to the initial value (Hoffmann et al., 2009). In the last decade,

besides chloride channels, studies have been focused on potassium channels as having an

important role in sensing the changes in cell volume and triggering regulatory volume

mechanisms. Activation of potassium current during volume changes has indeed been

reported in a great variety of cell types. These currents are transported by channels belonging

to distinct classes of K+ channels, the 6TM, 4TM or 2TM K+ channel family (See figure 2).

Interestingly, channels that are homologous have distinct behaviour with respect to cell

volume. Some are sensitive to volume and some are not (KCNQ1 vs. KCNQ2 or Slick vs.

Slack), probably indicating different cellular expression and functions.

Most of these potassium channels are regulated by instantaneous small changes in cell

volume as shown by Grunnet (Grunnet et al., 2003;Grunnet et al., 2002b). In these

experiments channels were expressed together with Aquaporin 1 (AQP1) in Xenopus oocytes.

Since oocytes are devoid of endogenous water channels, AQP1 was used as a “tool” to make

the oocytes swell or shrink. Oocytes exposed to osmotic challenges corresponding to a 27%

decrease in bath osmolarity showed an increase of approximately +8% in cell volume. A

reversible change in cell volume (-8%) was monitored upon a 27% increase in bath

osmolarity (Figure 4 B). These small changes in cell volume evoked dramatic current

responses. For instance KCNQ4 currents increased to 258% of control upon cell swelling

and decreased 30% of control upon cell shrinkage (Grunnet et al., 2003).


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Figure 2 Overview of volume sensitive potassium channels across the K+ family tree.

Blue: Volume sensitive K+ channels. Red: Homologeous K+ channels that are not volume sensitive.

Modified from (Coetzee et al., 1999;Goldstein et al., 2001)

The KCNQ1 channel

The experimental work in my thesis has been focused on KCNQ1 channel regulation by the

β subunit KCNE1 and by cell volume. The following paragraph will therefore mainly

concentrate on KCNQ1 and its regulation. KCNQ1, previously named KvLQT1, was the

first member of the KCNQ family (KCNQ1-5) to be cloned (Wang et al., 1996a). KCNQ1

channels belong to the 6 TMD family of K+ channels and have 4 positively charged amino

acids in the 4th TMD making them voltage gated. Four subunits assemble to make a

functional channel (Figure 3).

Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7

Kir4.1 Kir4.1-5.1

TWIK TREK TASK TRAAK THIK TALK KCNK

TASK1 TASK2

Eag KCNQKvKCa2+

eag elkerg

SKIKBK

Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9

Slo1 Slo3Slo2

Slick Slack

KCNQ1 KCNQ2 KCNQ3 KCNQ4 KCNQ5Kv1.3 Kv1.5

6TM

4TM

2TM


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Figure 3 Topology and channel architecture of KCNE and KCNQ1 proteins.

Left: Topology of the KCNE and KCNQ1 proteins with indications of some of the domains

important for regulation of the channel. Right: KCNQ1/KCNE channel architecture. Four KCNQ1

-subunits assemble to form the basic channel. KCNQ1 and KCNE coassemble in a 4:2

stoichiometry. (figure from (Jespersen et al., 2005).

1. Expression and role in epithelia and cardiac tissue

KCNQ1 channels have been found in a number of epithelial tissues and have been

demonstrated to be essential for transepithelial transport and for participating in potassium

absorption and secretion. In the inner ear, these channels play a role in maintaining the

proper ion balance needed for normal hearing. KCNQ1 and the auxiliary subunit KCNE1

are expressed in the marginal cells at the apical membrane of the stria vascularis in the

cochlea. These cells secrete the endolymph, which is a K+ rich fluid that bathes the stereocilia

of the sensory hairs cells and is a prerequisite for the sense of sound and balance. Any

mutations in KCNQ1 or KCNE1 leads to a low K+ concentration in the endolymph which

results in the degeneration of the sensory hairs cells in the auditory pathway and

consequently hearing and balance defects (Ashcroft, 2000;Bleich & Warth, 2000).

In other epithelia, such as pancreas, kidney and airway, KCNQ1 and KCNE1 are crucial for

providing a basolateral K+ conductance essential for driving apical Cl- secretion. This


20

important role was demonstrated in experiments where chromanol 293B specifically

inhibited KCNQ1 and further abolished Cl- secretion (Bleich & Warth, 2000). KCNQ1

together with KCNE2 have also been localized in the luminal membrane of gastric parietal

cells having a crucial role in gastric acid secretion (Heitzmann et al., 2004)

In cardiac tissue, KCNQ1 channels contribute to the repolarisation of the cardiac action

potential thereby recharging the muscle after each contraction to maintain a regular

heartbeat. Mutations in KCNQ1 gene give rise to long QT syndrome which is a cardiac

disorder that may cause arrhythmias, loss of consciousness and sudden death. It is

characterized by an abnormally long QT interval in the electrocardiogram (ECG) which

reflects the delayed repolarisation of the ventricular action potential. This prolonged action

potential can provoke a life-threatening arrhythmia called “torsade de pointes” where the

QRS wave changes continuously and swing up and down around the baseline in a chaotic

fashion.

Two forms of LQT have been described: an autosomal dominant form known as Romano-

Ward syndrome and a much rarer recessive form known as Jervall-Lange-Nielsen (JLN)

syndrome. Besides having cardiac abnormalities, patients with JLN syndrome also suffer

from deafness. This disease is also linked to mutations in KCNE1 β-subunit.

2. Regulation

KCNQ1 channel activity is regulated by many factors such as calcium, pH, protein kinases,

auxiliary β subunits and cell volume. In the following section I will only consider the two last

mentioned factors. However, the remaining will be mentioned along with the discussion.


21

Figure 4 Regulation of KCNQ1 by KCNE1 and cell volume.

A) When expressed alone, the KCNQ1 gives rise to a voltage dependent outward current that reaches

a steady state within 1s. Co-assembly of the regulatory β-subunit KCNE1 with KCNQ1 results in a

significant change of the electrophysiological properties of the channel. This induces a slowly

activating delayed rectifier current called IKs. The voltage activation threshold is shifted to a more

positive potential and the inactivation is almost completely absent. The KCNQ1/KCNE1 complex

contribute to the repolarisation of the cardiac action potential at the plateau phase (From Jespersen et

al., 2005) B) KCNQ1 channels expressed with AQP1 in Xenopus laevis oocytes challenged with a

hyposmolar or hyperosmolar extracellular solution . Oocyte cell volume (upper traces) and currents

(lower traces) were simultaneously measured. KCNQ1 currents increased to 172% of control upon

8% increase in cell volume and decreased 55% of control upon 8% decrease in cell volume. Co-

expression of KCNE1 significantly attenuated the swelling-induced increase in KCNQ1 current,

whereas the response to cell shrinking was unchanged. (From Grunnet et al., 2003).

a. Regulation by β subunits

KCNQ1 associates with accessory proteins encoded by the gene family of KCNE which

have overlapping tissue distribution with KCNQ1. These proteins, β subunits, are small with

A) Regulation by KCNE1

B) Regulation by cell volume


22

a single transmembrane domain (Figure 3). They do not produce current by themselves,

however, they change the actual function of the KCNQ1 channel and give it new

electrophysiological properties when they associate with it. Until now five different β

subunits for the KCNQ1 channel have been identified (KCNE1-KCNE5). These subunits

alter cell surface expression and modulate gating properties (Tinel et al., 2000;Angelo et al.,

2002;Grunnet et al., 2002a;Jespersen et al., 2005).

The association of KCNQ1 with KCNE1 is the most studied example as they form the

slowly activated delayed rectifier K+ current, IKs, which contributes to the repolarisation of

the cardiac action potential and any mutations in KCNQ1 or KCNE1 has been related to

LQT syndrome (Ashcroft, 2000).

KCNE1 considerably increase the KCNQ1 current amplitude, delay its activation,

inactivation and shifts the voltage dependence of activation (Figure 4 A). It is now well

established that KCNE1 lies in close proximity to the KCNQ1 pore and thereby influencing

KCNQ1 conducting properties and pharmacology. Studies have shown that KCNE1 directly

interacts with the S5-P-S6 pore domain and sits in a cleft between this pore domain and

adjacent voltage sensor (Kang et al., 2008;Panaghie et al., 2006;Melman et al., 2004).

Moreover, some residues from the KCNE1 transmembrane domain modulates channel

activation (Chen & Goldstein, 2007;Melman et al., 2002) whereas the juxtamembrane C-

terminal domain of KCNE1 is important in preventing channel inactivation (Chen et al.,

2009;Tapper & George, Jr., 2000).

Studies have shown that KCNE1 requires KCNQ1 co-assembly prior to reaching the cell

surface (Chandrasekhar et al., 2006;Vanoye et al., 2010) and newer studies reveal, that once

the subunits have been delivered to the membrane they can dissociate from each other

(Poulsen & Klaerke, 2007;Jiang et al., 2009) and that KCNQ1 can alternate between being

associated with KCNE1 and KCNE2 (Jiang et al., 2009).

Recently, another member of the one transmembrane segment protein family, the

corticosteroid hormone induced factor (CHIF) have been shown to be capable of

modulating KCNQ1 channel by making the channel constitutively open at all potentials but

so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue

is lacking (Jespersen et al., 2006).


23

b. Regulation by cell volume

Sasaki et al (Sasaki et al., 1994) were the first to report an increase in the slowly activating

current IKs, during exposure to cell swelling in guinea pig ventricular myocytes. Subsequently,

newer studies supported this finding and suggested the channel to contribute to the

regulatory volume response in similar and different cell types such as in canine ventricular

myocytes (Zhou et al., 1997), primary neonatal rat cardiomyocytes (Vandenberg et al. 1996;

Calloe et al. 2007), airway epithelial cells (Lock & Valverde, 2000), rat hepatocyte (Lan et al.,

2005), guinea-pig ventricular myocytes (Missan et al., 2006) and mammary epithelial cells

(vanTol et al., 2007). Volume-sensitivity of homomeric expressed KCNQ1 channels was also

demonstrated in Xenopus oocytes and in COS cells (Kubota et al., 2002;Grunnet et al., 2003).

This indicates that KCNQ1 activity upon volume changes is independent of KCNE1 and of

the expression system.

Grunnet et al., (2003) have demonstrated that the channel activity augments with increased cell

volume and decreased when the cell volume diminished. These cell volume changes where

within physiological ranges (8-10% volume increase or decrease) (Figure 4 B). Since it is well

documented that epithelial cells change volume during transport of salt and water, the property

of being a precise sensor of even small changes in cell volume may explain how the activity of

this otherwise “voltage regulated” K+ channel can be modulated in epithelia and play a

significant physiological role.

Here are some examples of the role of the volume sensitive KCNQ1 channels in

physiological and ischemic conditions:

Role of volume sensitive KCNQ1 in mammary epithelium

Mammary epithelial cells experience changes in volume as a result of variations in milk

metabolism and to the presence of the higher content of impermeable solutes such as lactose

in milk. Furthermore, the K+ concentration of milk is actually several fold higher than that of

plasma suggesting that some mechanisms for K+ secretion and volume regulation must be

present in mammary epithelial cells (Shennan & Gow, 2000). In 2007, KCNQ1 expression

has been reported for the first time in mammary epithelial cell line. KCNQ1 channels have

been exclusively localized at the apical membrane. By using both pharmacological (293B and

XE991) and molecular (heterologous expression of dominant negative ΔN-KCNQ1


24

construct) means, vanTol et al have demonstrated that inhibition of KCNQ1 activity

abolished the ability of MCF-7 cells to undergo RVD suggesting a physiological role for

KCNQ1 in regulating mammary epithelial cell volume (vanTol et al., 2007).

Role of volume sensitive KCNQ1 in liver cells

Hepatocellular nutrient uptake and bile formation in the liver is accompanied by cell swelling,

activation of K+ currents and subsequent RVD. Lan W-Z et al, have demonstrated that

KCNQ1 channels are indeed participating in the RVD-induced K+ efflux in intact liver.

Furthermore, PIP2 indirectly regulates swelling activated potassium current through a PLC-

dependent process involving PKC activation and cytoskeletal rearrangement (Lan et al.,

2006).

Role of volume sensitive KCNQ1 in cardiomyocytes

During ischemia and reperfusion, cardiomyocytes may experience significant cell swelling

due to the breakdown of high energy phosphates and macromolecules (e.g., glycogen, free

fatty acids) and the accumulation of lactate within the cell causing swelling. Cell swelling

induces activation of IKs current which promotes repolarisation and shortening of the cardiac

AP, which in turn may restrict Ca2+ influx and protect myocytes against deleterious Ca2+

overload. Calloe et al, have demonstrated that a slowly activating current mediated by the

KCNQ1 channels in complex with the beta subunit KCNE1 is activated in neonatal rat

cardiomyocytes upon cell swelling. This current was shown to contribute to the RVD

response after ischemia by the help of an intact F-actin cytoskeleton (Calloe K et al 2007).

The swelling induced IKs current may protect the cells from Ca2+ overload however the

shortening of the cardiac AP can lead to cardiac arrhythmias.


25

The following section is written as an introduction to purinergic receptors related to

manuscript II.

Purinergic receptors and ATP signalling

Extracellular ATP release is reported in various cell types, e.g. in neuronal cells acting as a

neurotransmitter, as well as in epithelial cells acting as an autocrine/paracrine messenger

modulating many cellular functions. This ATP release can be triggered by neuronal and

hormonal agonists (Abbracchio et al., 2009;Joseph et al., 2003;Novak, 2003) and by

mechanical stimuli such as shear stress (Woo et al., 2008;Grierson & Meldolesi, 1995;Maroto

& Hamill, 2001), compression (Sauer et al., 2000), stretch (Grygorczyk & Hanrahan, 1997)

and cell volume changes (Boudreault & Grygorczyk, 2004;Grygorczyk & Guyot, 2001;Aleu et

al., 2003). Once outside it interacts with purinergic receptors located at the cell membrane

and subsequently triggers distinct intracellular signalling pathways dependent on the receptor

type. Released ATP during cell swelling has been reported to activate ion channels such as

chloride and potassium channels and to contribute to the regulatory volume decrease (Wang

et al., 1996b;Roman et al., 1997;Feranchak et al., 2000;Perez-Samartin et al., 2000;Hafting et al.,

2006;Almaca et al., 2009).

Receptors

Purinergic receptors are divided into two families: 1) P1 receptors that recognize adenosine

and which are divided into 4 subtypes A1, A2A, A2B and A3. They are coupled to G proteins

and 2) P2 receptors that recognize ATP, ADP, UTP and UDP and which are divided into

two subfamilies: ionotropic P2X receptors, which are ligand gated ion channels, and

metabotropic P2Y receptors which are G-protein coupled (Figure 5).

Binding of an agonist to the extracellular loop of P2X receptors will mediate receptor

conformational change resulting in a rapid non selective transport of cations (Na+, K+, Ca2+)

across the cell membrane. A subsequent increase in intracellular calcium elicits membrane

depolarization and activation of calcium dependent processes.

Adenosine and P2Y receptors are G-protein coupled receptors that couple to 1) Gi/Go

proteins which inhibit adenylate cyclase (AC) and subsequently decrease cAMP, 2) Gs

proteins which activate AC and increase cAMP or 3) Gq proteins which activates membrane

bound phospholipase C (PLC) that hydrolyses Phophatidylinositol 4,5 biphosphate (PIP2)


26

into inositol triphosphate (IP3) and diacyglycerol (DAG). IP3 will help calcium release from

calcium channels at the ER membrane and DAG will lead to activation of protein kinase C

which phosphorylates many other proteins.

Figure 5 Overview of purinergic receptors.

ATP is released to the extracellular space either by exocytosis or with the aid of membrane

transporters. Released ATP acts on P2X and P2Y receptors. P2X receptors which are ligand gated

ion channels consists of three subunits that allow the passage of cations. P2X receptors are encoded

by seven distinct genes (P2X1 to P2X7). P2Y receptors are proteins consisting of 7 transmembrane

domains where the C-terminal is coupled to an intracellular G-protein. Two distinct subgroups of

P2Y receptors are recognized dependent on the G-protein they couple to. The P2Y1, P2Y2, P2Y4,

P2Y6 and P2Y11 subgroup use Gq and the P2Y12, P2Y13 and P2Y14 subgroup couple to Gi/o

protein. ATP is hydrolyzed by membrane bound ecto-nucleotideases (NTPDase: ecto-nucleoside

triphosphate diphosphohydrolases) to ADP and AMP. ADP acts also on P2Y receptors. 5-

nucleotidase (5'-NT) catalyses the hydrolysis of AMP to adenosine which activates P1 receptors

which are also G-protein coupled receptors. (Figure taken from http://www.uni-

leipzig.de/~straeter/research/ntpdase.html).

ATP release mechanisms

Under basal conditions, intracellular concentration of ATP lies between 1-5 mM.

Extracellularly, ATP concentrations are regulated due to the action of membrane bound

ecto-nucleotideases, enzymes that degrades ATP. This concentration gradient favours the


27

movement of ATP out of the cell. Several mechanisms have been suggested regarding how

ATP is released under basal and stimulated conditions. It can be released by a non-ionic

process through mechanical induced constitutive release of vesicles (Maroto & Hamill, 2001)

and through hormonal/neuronal regulated exocytosis (Lazarowski et al., 2003). Moreover

release can take place by a conductive ionic process through membrane channels, such as

mechanically gated ion channels (Aleu et al., 2003), hemichannels such as connexins (Bahima

et al., 2006) and pannexins (Huang et al., 2007), maxi anion channels (Liu et al., 2008), volume

regulated anion channels (Hisadome et al., 2002;Fitz, 2007), CFTR (Schwiebert et al., 1995)

and P2X7 receptors (Suadicani et al., 2006).

When studying the role of ATP in regulatory volume decrease or increase, some classical

tests are used such as looking upon the effect of exogenously added ATP, purinergic

receptor blockers or agents that degrade released ATP (such as apyrase) on the cell volume

induced currents. Some of these methods were also used in one of the studies in this thesis

(manuscript II).


28

THESIS OBJECTIVES

The overall objective of the whole study was to identify the different mechanisms underlying

KCNQ1 sensitivity upon changes in cell volume.

The detailed aims of this thesis were:

1) To test whether potassium channel sensitivity upon changes in cell volume is mediated

through sensitivity to changes in membrane stretch (Manuscript I).

We intended to differentiate between membrane stretch and cell volume and examined

whether these phenomena share a common mechanism or they are different on their way

affecting ion channel regulation. This was examined by the use of the patch clamp

technique on different ion channels that have been reported to be activated by one of the

two mechanisms (the volume sensitive KCNQ1 channel and the stretch-sensitive BK

channel).

2) To test whether volume-activation of KCNQ1 channels could be mediated by an

autocrine mechanism in which ATP released in response to volume changes activates

signalling pathways that subsequently lead to ion channel stimulation (Manuscript II).

Several studies have shown that ATP released during mechanical stimuli for example

upon changes in cell volume has an important role in cell volume regulation and

modulating ion channel activity. The goal of the study was first, to monitor ATP release

under basal conditions and during cell volume changes for the KCNQ1 injected oocytes

and second, to investigate whether ATP release modulates KCNQ1 cell volume

sensitivity and to examine the effect of addition or removal of ATP from the

extracellular side.

3) To determine whether expression of KCNQ1 with the KCNE1 alters the number of ion

channels translocated to the membrane (Manuscript III)

It is debated whether the increase in macroscopic current upon expression of KCNQ1

with KCNE1 is due to an increase in ion channel conductance (γ), the open state

probability (Po) or an increase in the number of channels in the plasma membrane (N).

The latter was quantified by measuring the level of KCNQ1 surface expression by using

an enzyme-linked immunoassay.


29

METHODS

In the following section, the techniques used during the thesis will be briefly presented. More

details related to the specific experiments are described in the different manuscripts.

In this project, oocytes from the aquatic adult female frog Xenopus laevis are used for the

heterologous expression of ion channels. The frogs are supplied from the animal facility at

the August Krogh Building or the Panum Institute where they are bred under ideal

temperature and light conditions. Collagenase treated and defolliculated oocytes are also

purchased from the German company Ecocyte. Xenopus oocytes have a developed apparatus

for synthesis of foreign injected protein meanwhile lowering the production of their own

endogenous proteins (Dascal, 1987). Many electrophysiological techniques can be applied on

oocytes for example two-electrode voltage clamp (TEVC), cut open, patch clamp and

macropatch or giant patch technique.

Two-Electrode voltage clamp technique (TEVC)

Because of its large size compared to other cells (e.g. CHO, HEK cells), Xenopus Oocyte

whole cell currents can not be measured with the conventional patch clamp technique at the

whole-cell configuration. The surface area of an oocyte is large with invaginations; therefore

there is an enormous amount of membrane that must be charged in order to clamp the

oocyte. Therefore the TEVC is a straightforward technique to measure whole cell currents

on Xenopus oocytes without any effect or change on the intra-oocyte concentration.

Recordings can be made directly on oocytes still coated with the vitelline membrane and

experiments can be repeated up to a week after if the oocytes survive that long.

As the name refers to, the technique consists of two electrodes of very thin tips which are

inserted into the oocyte. One intracellular electrode is used to record the actual intracellular

potential (the voltage electrode) and the second electrode is used to pass current in such way

as to maintain the desired potential (the current electrode). This is achieved using a feedback

circuit. The principle of the technique is to inject a current which is equal in amplitude but

opposite in sign to that which flows across the cell membrane. This technique measure the

current that flows through the whole cell membrane. This whole cell current is the sum of

the currents flowing through several different kinds of ion channel in the oocyte


30

The recording chamber is connected to a perfusion system, which make it easy to apply

different solutions for example employing solutions with different osmolarities in order to

make cell volume experiments meanwhile measuring current alterations.

The patch clamp technique

The patch clamp technique is a method that allows direct observation of single-channel

activity thereby deducing the single-channel conductance and channel kinetics. Moreover

whole-cell currents from small cells are also recorded. The technique is based on making a

high resistance seal, a so called giga seal between the glass pipette tip and the cell plasma

membrane by applying a negative pressure in the pipette. This high resistance seal enable us

to record small amplitude of single-channel in the order of pA at very low level of

background current noise. This technique uses a single electrode both to control the

membrane potential and to measure currents.

An advanced method in patch clamping which is commonly used for bigger cells such as the

Xenopus laevis oocytes or muscle cells is the so called macropatch or giant patch technique

(depending on how big a pipette tip is used). This method has been developed by Hilgemann

D. W in 1989 on cardiac myocytes (Hilgemann, 1989). It is basically a cell-attached

configuration that allows measuring macroscopic currents from much larger membrane areas

containing hundreds of ion channels than in conventional single-channel recording. This

requires large diameter patch pipettes of approx. 5-30 μm in tip diameter and a resistance of

a few hundred kiloohms. The advantage of this method is that it allows macroscopic current

measurements with high signal to noise ratio and because of the low access resistance,

microsecond time resolution can be achieved.

ATP bioluminescent assay

In Manuscript II, Basal ATP release and release during cell volume changes is measured by

ATP bioluminescent assay based on the firefly Luciferase. This is a widely used tool for

conducting quantitative ATP measurements. The assay is based on the following reaction:

ATP is consumed and light is emitted when firefly luciferase catalyzes the oxidation of D-

luciferin:

Firefly luciferase

ATP + Luciferin Adenyl-luciferin + PPi

Mg++

Adenyl-luciferin + O2 Oxyluciferin + AMP+ CO2 + Light


31

The first reaction is reversible and the equilibrium lies far to the right. The second reaction is

essentially irreversible. When ATP is the limiting reagent, the light emitted is proportional to

the ATP present.

Enzyme linked immunoassay for surface expression

In Manuscript III, the surface expression of KCNQ1 channel was studied by means of

enzyme linked immunoassay. This was conducted on a double Hemaglutunin (HA) tagged

KCNQ1 channel. The two HA-epitopes, with the amino acid sequence YPYDVPDYA, are

placed in the extracellular site between the S3 and S4 segment of the KCNQ1 protein. This

allows us to measure the amount of surface expressed protein through enzyme immunoassay

(Figure 6).

Figure 6 Measurement of surface expressed proteins through enzyme immunoassay.

The extracellular segment between S3 and S4 of KCNQ1 is tagged with two HA epitopes. If

KCNQ1 is expressed on the cell surface, the HA epitope is accessible to the primary anti-HA

antibody. In the second step, bound primary antibody is recognized by a HRP-conjugated secondary

antibody. Bound HRP reacts with the OPD solution and gives a yellowish color which can be

quantified measuring the absorbance at 450 nm.

S1 S2 S3 S4 S5 S6

N C

Extracellular

Intracellular

Color

OPD

substrate

HA-tagged protein complex

Anti HA primary antibody

HRP- conjugated secondary antibody

Absorbance at 450 nm


32

RESULTS AND DISCUSSION

Cells have several ways to accommodate for volume expansion and for preventing cell lysis

when exposed to hypoosmotic conditions. Both physical changes related to the membrane

and the cytoskeleton and intracellular changes that restore optimal intracellular

concentrations of enzymes, osmolytes and metabolites occur in order to bring back normal

cell volume. Potassium channels play a critical role in regulating cell volume as they are able

to “sense” the changes in cell volume and subsequently having a regulatory effect on it.

Several mechanisms for coupling of cell volume changes and K+ channel activation have

been proposed and are still under discussion.

This thesis has focused on identifying the mechanisms underlying the volume sensitivity of

KCNQ1 channel. KCNQ1 surface expression upon interaction with the regulatory subunit

KCNE1 was also studied. The main findings are:

1. Cell swelling and membrane stretch are two independent regulatory mechanisms and

i.e. KCNQ1 cell volume sensitivity is not mediated by membrane stretch (Figure 4 in

manuscript I)

2. KCNQ1 channel response to cell volume changes is not mediated by ATP release

(Figure 3 and 4 in manuscript II)

3. KCNE1-induced increase in KCNQ1 currents is not mediated through enhanced

plasma membrane expression (Figure 3 in manuscript III)

In the following discussion, these results will be discussed in line with other possible

mechanisms. Where it is appropriate, I have attempted to propose different perspectives

related to the further identification of the mechanisms underlying channel activation upon

changes in cell volume.


33

Cell swelling vs. membrane stretch

Activation of ion channels by membrane stretch and by cell volume changes have been

considered until recently as one common mechanism. The study in Manuscript I, discloses

that volume changes and membrane stretch are two distinct mechanisms. Ion channel

activation during cell swelling seems not be a result from membrane stretch and even though

a channel is activated by stretch, it does not seem to react upon cell swelling. This suggests

that stretch and swelling may activate K+ channels by distinct mechanisms and that they do

not depend on each other. From this finding we can conclude that KCNQ1 cell volume

sensitivity is not mediated by membrane stretch.

If the stretch sensitive BK channels act as a “biosensor” of cell membrane stretch and if

swelling stretches the membrane then a BK response would be elicited. Whether cell volume

changes is in fact a membrane phenomenon and whether membrane stretch really takes place

during cell swelling have been of major debate. Groulx 2006 has shown that during moderate

swelling (50% decrease in osmolarity) cells increase their surface area by 30% mainly by

unfolding the surface membrane. In the other hand, under extreme hypotonic swelling (98%

decrease in osmolarity) the cell prevents lysis by exocytotic insertion of membrane from

intracellular pools or by stretching the membrane. However, in most physiological situations,

Groulx indicates that membrane tension is unlikely to reach the level required to activate

stretch regulated ion channels (Groulx et al., 2006).

Earlier studies have shown that stretch-induced activation of some K+ channels found at the

single channel level is confirmed at the whole cell level through the increase of K+ currents

in hypotonic solutions (Sackin, 1989;Filipovic & Sackin, 1992;Davidson, 1993;Allard et al.,

2000;Christensen & Hoffmann, 1992). However, we have to remain critical to these results

as in these studies, cells were exposed to more than 50% reduction in bath osmolarity (a

hypotonic shock) resulting in an increase in cell volume to e.g. 66% (Sackin, 1989) whereas

Grunnet et al. (2003) showed that oocytes exposed to osmotic challenges of -50 mOsm/l

(27% reduction in bath osmolarity) increased about +8 % of the volume, which is much

more closer to physiological ranges during “real life”. In fact, the use of unphysiologically

large hypoosmotic shock may optimize the chance of seeing changes in mechanosensitive

channel activity as a last line of defence against excessive cell swelling. Grunnet et al., 2003

considered KCNQ1 channels as precise sensors because they sense very small changes in

volume. That means, theoretically speaking, that a hypoosmotic shock (>50% decrease in


34

osmolarity or more) that will provoke membrane tension due to unfolding and smoothing

out of excess membrane area of the oocyte (in the form of microvilli and membrane folds) is

not necessary to activate KCNQ1 channels, but very small volume changes are enough to

activate them. This again points out that KCNQ1 sensitivity to these very low changes in cell

volume is not membrane-mediated, but could be due to cell volume induced alteration of

intracellular components, e.g. second messengers that subsequently affected the activity of

the channels.

In another recent study, atomic force microscopy made it possible to “picture” the effect of

cell volume expansion on the plasma membrane (Spagnoli et al., 2008). Unexpectedly, the

study has shown that the cells get softer and not stiffer when they are swollen; hence no

membrane stretch is taking place. This is due to the sponge like property of the cytoskeleton

which bears most of the osmotic stress whereas little is attributed to the membrane.

From our study and the study by Spagnoli, we have proven that cell volume changes may not

be confined to the cell membrane tension per se and hence the volume response of KCNQ1

is mediated through an alternative mechanism.

A recent study (Otway et al., 2007), reports a novel volume-responsive KCNQ1 mutation in

a kindred with late-onset familial atrial fibrillation. The variant called R14C on the KCNQ1

gene having a cysteine instead of an Arginine at codon 14, is located within the short

cytoplasmic N-terminus of the KCNQ1 protein. Functional studies have indicated a more

marked response to cell swelling compared to wild type IKs channels: a higher increase in

current, a more leftward shift in the voltage dependence of activation, faster acceleration of

activation and slowing of deactivation were observed. This variant had a gain of function

effect causing cardiac action potential shortening (Otway et al., 2007). However in this study

they named this variant a stretch sensitive KCNQ1, though based on cell volume

experiments and not on membrane stretch. Yet, it could be interesting to expose this R14C

variant to real membrane stretch through pipette pressure in order to confirm or reject this

supposed stretch sensitivity.

The key finding in manuscript I, is that membrane stretch and cell volume changes constitute

independent ion channel regulators. This is true for KCNQ1 and BK channels used in the

study. Whether the assumption is a general property relating to all mechanosensitive ion

channels should be further examined. For this purpose e.g. the TRAAK and TREK channels

belonging to the two-pore potassium channel family are particularly interesting, since they


35

are apparently sensitive to small changes in cell volume as well to membrane stretch (Patel et

al., 2001). In addition, it seems well documented that the stretch sensitivity of the TREK

channels is dependent on a single amino acid, namely a positively charged amino acid located

in close proximity to the cell membrane at the inner part of transmembrane segment 2

(Honore et al., 2002). It would be of great interest to change the charged amino acid to a

neutral amino acid by site directed mutagenesis, a procedure which has earlier been shown to

eliminate the stretch sensitivity, and subsequently expose the mutated channels to cell

volume changes and membrane stretch. If the channel is still volume sensitive then this will

confirm our conclusion that cell volume and membrane stretch are two mechanisms

regulating the same channel type by two different ways.

ATP release and cell volume changes

Many studies have shown that release of ATP induced by cell swelling can modulate chloride

and potassium channels through G-protein coupled signalling pathways (Wang et al.,

1996b;Roman et al., 1999;Perez-Samartin et al., 2000;Light et al., 2003;Darby et al.,

2003;Hafting et al., 2006). In the case of the volume sensitive potassium channel KCNQ1, we

demonstrated in manuscript II that released ATP upon changes in cell volume in

KCNQ1±AQP1 injected oocytes does not contribute to the volume sensitivity of KCNQ1

channels indicating that ATP does not modulate KCNQ1 activity through a purinergic

signalling pathway in our expression system.

Interestingly, previous studies have shown a coupling of the KCNQ1 and its auxiliary β-

subunit KCNE1 to purinergic signalling in native tissues. The strial marginal cells and

vestibular dark cell of the inner ear of rodents expresses both KCNQ1/KCNE1 and

purinergic receptors (P2Y4) at the apical membrane having an important function in

endolymph homeostasis and protection from overstimulation (Housley et al., 2009;Lee &

Marcus, 2008). KCNQ1/KCNE1 channel activity and thus K+ secretion was shown to be

modulated by 3 purinergic pathways in rodents when P2Y4 receptors are stimulated: 1) G

protein-PLC activation leads to consumption of membrane PIP2 with the consequent

reduction of K+ channel activity; 2) the DAG-PKC path decreases K+ channel activity

directly via phosphorylation of the channel; and 3) the IP3/Ca2+ path decreases the channel

activity directly via the effect of Ca2+ on channel activity (Lee & Marcus, 2008). Additionally,

Honoré et al (1992) have reported that stimulation of purinergic receptors regulates the

KCNQ1/KCNE1 channel activity in mouse heart. This study was concluded by injection of


36

cardiac polyA+ RNA from neonatal mouse heart into Xenopus oocytes. The RNA directed

the expression of IKs channel as well the expression of purinergic P2 receptors. By

stimulating these receptors it produced intracellular Ca2+ increase, DAG and thereby

activating protein kinase C which alters IKs activity (Honore et al., 1992).

It seems obvious to consider a similar purinergic signaling mechanism taking place during

changes in cell volume. Yet, extracellular added ATP that activate Gq receptors and stimulate

PKC activity did not have any effect on the stimulation of IKs current by hypoosmotic

solution in guinea-pig ventricular myocytes (Missan et al., 2006).

The fact that we did not see any coupling of KCNQ1 channel during volume changes with

purinergic signaling may be because there is indeed no link, or because of the expression

system that we used. We cannot exclude that some components necessary to initiate or

complete the purinergic signaling pathway are missing in the Xenopus oocyte expression

system that are normally present in warm blooded animal cells. It may be therefore necessary

to try in another expression system for example HEK cells or COS cells. Moreover, we are

not sure that purinergic receptors are present in defolliculated oocytes (Discussed in

manuscript II); however adenosine receptors are apparently present. Whether a signaling

pathway via adenosine receptor can take place should be investigated. Moreover, according

to the previous studies in native cells KCNE1 is implicated in the functional purinergic

signaling pathway. In our study we did not include KCNE1. We may therefore need to

repeat the same experimental protocol with KCNQ1/KCNE1 coexpression.

KCNQ1 association with KCNE1 and volume sensitivity

Besides being volume regulated, KCNQ1 is regulated by the β subunit KCNE1. Heteromeric

association of KCNE1 with KCNQ1 induces a current with electrophysiological channel

properties markedly different from that of the KCNQ1 channel itself. KCNE1 association

give rise to a much larger current than the one seen by KCNQ1 alone. This increase in

current is not mediated by an increase in the number of ion channels translocated to the

membrane (N) as shown in Manuscript III however it may be due to an increase in the open

state probability (Po) or the single channel conductance (γ). These findings are in agreement

with previous studies which additionally show an increase in ion channel conductance upon

coexpression (Yang & Sigworth, 1998;Pusch, 1998;Sesti & Goldstein, 1998).


37

In relation to cell volume experiments, there is conflicting results as to whether KCNE1 is

crucial for the volume sensitivity of KCNQ1. Studies on proximal convoluted tubule

epithelial cells from KCNE1 knockout mice, show that KCNQ1 failed to respond to cell

swelling, indicating that KCNE1 is essential for KCNQ1 volume sensitivity (Lock &

Valverde, 2000) However, other studies indicate that the volume sensitivity is exclusively a

KCNQ1 property, though slightly modulated by KCNE1 (Grunnet et al., 2003;Kubota et al.,

2002) (Figure 4 B).

In principle, KCNQ1 channels could respond to cell volume changes by modulation of

either (i) their open state probability, (ii) their single channel conductance, or (iii) by changes

of the number of channels inserted in the plasma membrane. In Manuscript III, we used an

enzyme-linked immunoassay and a HA-tagged version of the KCNQ1 channel in order to

estimate the number of ion channels present in the plasma membrane in the presence and

absence of KCNE1. This method is a plausible way to measure the number of ion channels

at the cell membrane during resting and during swelling or shrinkage. For this purpose,

oocytes need to be fixed at the different osmolarities, however preliminary trials with the

formaldehyde as a fixative made the oocytes to shrink. Since the goal was to measure the

number of ion channels during cell swelling and cell shrinkage, we could not proceed with

this method.

Groulx postulated that exocytotic insertion only takes place under extreme hypoosomotic

challenges; it seems that KCNQ1 current increase during modest cell swelling is then not due

to alteration in the number of ion channels in the membrane surface. However, this can be

further elucidated by the use of laser based total internal reflection fluorescence microscopy,

a technique that visualizes events happening at the membrane surface such as vesicle

trafficking and subsequent fusion and release of individual ion channels (which are

fluorescently labelled) at the plasma membrane. This should allow us, in real time, to detect if

channels are moved in and out of the plasma membrane of the Xenopus oocyte during cell

volume changes.

Recently a potassium channel belonging to the BK family called Slick (Slo2.1) was shown to

be volume sensitive (See table 1 and figure 2). This channel has a high conductance and

therefore single channel events are easily detected with the patch clamp technique unlike the

KCNQ1 channel. It would therefore be interesting to conduct single channel recordings on

the slick channel in order to look after any alteration in Po or single channel conductance

during changes in cell volume.


38

The cytoskeleton

A rearrangement of the underlying cytoskeleton occurs during cell volume changes and in

particularly actin filaments have been implicated in the modulation of ion channels during

cell swelling and shrinkage (Pedersen et al., 2001). Treatment with cytochalasin D, which

interferes with actin assembly has shown inhibition of KCNQ1 current in Xenopus oocytes

(Grunnet et al., 2003) and in cardiomyocytes (Calloe et al., 2007) and subsequent abolishment

of the regulatory volume decrease. This indicates that an intact actin cytoskeleton is

important for activation of channels by cell swelling. For the KNCQ1 channels there is

evidence that the interaction with the cytoskeleton takes place at the N-terminal of the

channel protein.

In contradiction, similar studies where guinea-pig ventricular myocytes were treated with

cytochalasin D had no effect on the response of IKs to hypoosomotic solution (Missan et al.,

2006). Additionally, the volume sensitive Slick channel was not affected either by

Cytochalasin treatment in Xenopus oocytes (Stolpe K. and Tejada M., personal

communication). Therefore, the cytoskeleton may in some cases be involved in cell volume

regulation of K+ channels, but it does on the other hand not seem to be a crucial player.

Intracellular calcium

It has been suggested that KCNQ1 can be inhibited (Shen & Marcus, 1998) or augmented

(Kerst et al., 2001) by intracellular Ca2+, or indeed wholly Ca2+ insensitive unless coexpressed

with KCNE1 (Boucherot et al., 2001). Apparently, a change in cytosolic calcium can not be

detected in Xenopus oocytes during volume changes (Vandorpe et al., 1998;Grunnet et al.,

2002b). Moreover, in rat hepatocytes and colonic epithelial cells volume induced IKs current

was shown to not be dependent on an increase in intracellular calcium (Lan et al., 2005)

(Kunzelmann et al., 2001)

Cytosolic pH

There is evidence that upon cytosolic acidification, homomeric KCNQ1 channel current is

inhibited whereas heteromeric KCNQ1/KCNE1 channel current is activated (Unsold et al.,

2000;Heitzmann et al., 2007). In Xenopus oocytes a significant change in cytosolic pH


39

apparently does not occur upon the small changes in cell volume. Previous data have shown

that the almost pH-insensitive Kir 4.1 and the strongly pH-sensitive Kir.4.1-Kir 5.1

responded almost identically to cell volume changes when expressed in Xenopus oocytes

indicating no alteration in pH (Soe et al., 2009). It seems therefore unlikely that KCNQ1

volume response is due to changes in intracellular pH. However, this may be different in

other expression systems or native cells. In fact, cardiac muscle tissue gets acidified down to

a pH of 5.5-6.5 during heart ischemia and the fact that KCNQ1/KCNE1 current activates

during acidification may have an important pathophysiological function (Heitzmann et al.,

2007).

Membrane PIP2

A number of studies suggest a role of the phospholipid PIP2 as an important regulator of K+

channels (Hilgemann, 1997) among them are the Kir channels (Logothetis et al., 2007) and

KCNQ channels (Zhang et al., 2003) (Lan et al., 2005;Loussouarn et al., 2003;Park et al.,

2005). Hepatocellular volume current mediated by KCNQ1/KCNE1 has been shown to be

dependent on PIP2 (Lan et al., 2005;Loussouarn et al., 2003;Park et al., 2005).

The requirement of PIP2 for KCNQ1/KCNE1 activity raises the possibility of physiological

regulation of the channel complex by receptor coupled PLC. This evidence is provided for

vestibular dark cell of the inner ear where purinergic receptor signalling has been coupled to

the modulation of KCNQ1/KCNE1 channel through a G protein-PLC pathway (Lee &

Marcus, 2008) earlier mentioned.

Upon volume increase and decrease in Ehrlich ascites cells, a respective decrease and

increase in membrane PIP2 was observed (Nielsen et al., 2007). Given that

KCNQ1/KCNE1 activity decreases upon consumption of PIP2 shown in earlier studies

(Lee & Marcus, 2008;Matavel & Lopes, 2009), the increase in KCNQ1 activity that we see

during cell swelling in Xenopus oocytes does not coincide with a PIP2 regulation.

Kinases

Protein kinase A and C are also known to be modulators of KCNQ1 channels (Boucherot et

al., 2001;Grunnet et al., 2003;Schroeder et al., 2000;Kunzelmann et al., 2001). An earlier study,

reinvestigated the possible involvement of PKA, PKC and Tyrosine kinase in the


40

hypoosmotic stimulation of cardiac IKs in guinea-pig ventricular myocytes. They

demonstrated that neither PKA, PKC (also in agreement with Grunnet 2003), PKG nor PI3-

K is mediating KCNQ1/KCNE1 cell swelling response. However, Tyrosine kinase did have

an effect (Zhou et al., 1997;Missan et al., 2006;Missan et al., 2008) coinciding with the

presence of 7 tyrosine phosphorylation sites on KCNQ1 (Missan et al., 2006).

Specific residues for the volume sensitive potassium channels

At present, sensitivity to small changes in cell volume can be assigned to several potassium

channels. This is a new regulatory mechanism along with voltage gated and ligand gated

potassium channels. Whether this observable phenotype is related to a specific genotype and

whether these potassium channel proteins have common conserved/specific residues for

volume sensitivity is just beginning to be revealed. For the case of KCNQ1, it seems obvious

that the specific residue responsible for the channel volume sensitivity lies within the N-

terminal site. When the N-terminal end of 95 AA is deleted, the channel loses its ability to

respond to changes in cell volume (Grunnet et al., 2003). Additionally, the mutated variant

R14C on the KCNQ1 gene located within the cytoplasmic N-terminus was demonstrated to

cause a marked increase in activation in response to cell swelling compared to wild type IKs

channels (Otway et al., 2007). We have preliminary evidence that the 17 amino acids closest

to the cell membrane are crucial (personal communication with Dan A. Klærke). By

extensive mutagenesis in this region, it will be possible to determine exactly which amino

acids are involved.

As mentioned in the introduction, we have recently found that two other, homologous

channels, slo2.1 (Slick) and slo2.2 (Slack) show different responses to changes in cell volume,

Slick is sensitive, whereas Slack is not (Figure 2). A closely related potassium channels to

KCNQ1, the KCNQ2 does also exert non-volume sensitivity (Figure 2). This situation

provides us the opportunity to construct chimeras between KCNQ1 and KNCQ2 channels

or Slick and Slack, co-express the chimeras with AQP1 in Xenopus oocytes, and test their

ability to be regulated by changes in cell volume in order to identify what specific region in

the channel that is responsible for the volume sensitivity. These studies might help us to

identify a sequence motif that might be common to all volume regulated potassium channels.


41

Figure 7 Possible mechanisms for KCNQ1 cell volume sensitivity


42

CONCLUSION

Figure 7 summarizes the above discussed possible mechanisms for volume sensitivity of

KCNQ1 channel. These events may also be true for other potassium channels.

By looking at the figure, cell swelling seems to be an unspecific stimulus. Many processes can

take place and affect the membrane embedded ion channels. Physical changes of the

membrane such as membrane stretch, unfolding of membrane invaginations or insertion of

extra membrane reserves may occur, as well as the rearrangement of the underlying

cytoskeleton. Alteration in the intracellular calcium concentration and pH, ATP release,

activation of purinergic signalling cascades and diverse kinases are some of the many

processes that takes place modulating ion channel activity. These processes can vary from

one cell type to another, having different effect on a specific type of channel. They can be

interacting events occurring simultaneously, so interpreting cause and effect regarding the

volume sensitivity of a particular ion channel can be difficult. However, we can only

understand the mechanism by breaking it down to simpler components which are much

manageable to study. For the case of KCNQ1 channel, we have come to the conclusion that

membrane stretch and ATP release are not mediating cell volume sensitivity of the channel,

at least in our expression system. However, it became clear that we need to look into the N-

terminal end of the channel which is in contact with the actin cytoskeleton in order to

identify the specific sequence motif that might be responsible for the regulation of the

KCNQ1 channel by small changes in cell volume.


43

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56

APPENDIX

1 published article, a related press article and 2 manuscripts are included in the thesis:

I. Hammami S, Willumsen NJ, Olsen HL, Morera FJ, Latorre R, & Klaerke DA

(2009). Cell volume and membrane stretch independently control K+ channel activity.

J Physiol 587, 2225-2231.

II. Hammami S., Willumsen NJ, Klaerke DA, & Novak I. (2010). KCNQ1 channel

response to cell volume changes is not mediated by ATP release. (about to be

submitted)

III. Hammami S, Klaerke DA & Willumsen NJ (2010). KCNE1-induced increase in

KCNQ1 currents is not mediated through enhanced plasma membrane expression

(submitted)

Related paper: Cell swelling and membrane stretch – A common trigger of potassium

channel activation?


57

Manuscript I: Cell volume and membrane stretch independently control

K +

channel activity


58


59


60


61


62


63


64

Manuscript II: KCNQ1 channel response to cell volume changes is not

mediated by ATP release

Hammami S.1, Willumsen NJ

1, Klaerke DA

2, & Novak I

1.

1Department of Biology, Faculty of Science, University of Copenhagen, Denmark;

2 Department of Physiology and Biochemistry, IBHV, Faculty of Life Sciences, University of

Copenhagen, Denmark


65

Abstract

Objective: A number of K+ channels are regulated by small, fast changes in cell volume.

The mechanisms underlying cell volume sensitivity are not known. One frequently stated

hypothesis is that cell volume sensitivity is mediated by membrane stretch. In a recent

study we presented evidence against this assumption by showing that the highly volume

sensitive KCNQ1 channel is not affected by membrane stretch. Another hypothesis is that

cell volume could be mediated by an autocrine or paracrine mechanism in which ATP

released from the cells in response to volume changes activates signaling pathways that

subsequently lead to ion channel stimulation. Our aim was to investigate whether volume

sensitivity of KCNQ1 is dependent on ATP release. Methods: KCNQ1 K+ channels

were co-expressed with AQP1 in Xenopus laevis oocytes and currents were measured by

TEVC. Oocytes were subjected to volume changes by exposure to iso-, hypo- or

hypertonic media with and without application of ATP. In other experiments the

ATP/ADP hydrolyzing enzyme apyrase was added. ATP release was also confirmed by a

luciferin-luciferase assay in non-injected and KCNQ1±AQP1 injected oocytes before and

after swelling and shrinkage. Results: In electrophysiological experiments it was shown

that apyrase (7 U/ml) decreased all currents by about 50%. When oocytes were swelled or

shrinked, the relative increase or decrease in current was not affected by apyrase nor

application of extracellular ATP. Luminescence assay showed that there was an increase

in ATP release in response to mechanical and hypotonic/hypertonic stimuli. Basal ATP

release was also higher for the KCNQ1±AQP1 injected oocytes compared to the non-

injected. Conclusion: Based on our data to date, we postulate that KCNQ1 does not seem

to be responsive to ATP nor apyrase during cell volume changes in AQP expressing

oocytes. This indicates that purinergic signaling is not involved in volume

sensitivity/regulation of KCNQ1 channel.


66

Introduction

Adenosine triphosphate (ATP) is a highly hydrophilic molecule responsible for energy

storage inside the cells. In the last decades many studies have reported a continuous basal

release of ATP into the extracellular medium where it can function as an

autocrine/paracrine signal. Extracellular ATP interacts with purinergic receptors (ATP

receptors) located at the cell membrane and modulates many cellular functions, such as

regulation of tissue blood flow, growth, neuronal activity, epithelial transport and

response to pathogens (Corriden & Insel, 2010). Various cell types, both secretory and

non-secretory, have been shown to release ATP upon activation by neuronal and

hormonal agonists (Abbracchio et al., 2009;Joseph et al., 2003;Novak, 2003), as well

when exposed to mechanical stress and changes in cell volume. These studies also

indicate an important role of ATP in cell volume regulation (see review (Franco et al.,

2008).

It is well known that a number of K+ channels are regulated by small, fast changes in cell

volume and are involved in cell volume regulation (Jensen et al., 2005;Jorgensen et al.,

2003;Grunnet et al., 2003;Grunnet et al., 2002;Hougaard et al., 2004). Many theories

have been proposed on how they are triggered during cell volume changes. A possible

mechanism would be that volume-activation of ion channels could be mediated by an

autocrine/paracrine mechanism in which the induced ATP release in response to cell

volume changes activates signaling pathways that subsequently lead to ion channel

stimulation as previously shown for different other ion channels (Hafting et al.,

2006;Franco et al., 2008;Corriden & Insel, 2010).

In our laboratory, ion channel activity during small and fast changes in cell volume is

studied by coexpressing potassium channels with AQP1 in Xenopus oocytes, which are


67

natively devoid of water channels. Grunnet et al (2002) have shown that in non-AQP1

expressing oocytes, decreasing or increasing the osmolarity of the extracellular medium

by 50 mOsm, changed cell volume by less than 0.2% after 50 s. In contrast oocytes

expressing AQP1, they responded immediately with significant changes in volume; these

oocytes swelled or shrank, respectively, by approximately 5% within 50 s and volume

continued to change for more than 300 s (Grunnet et al., 2002). Particularly, the

potassium channel KCNQ1 is very sensitive to changes in cell volume (Grunnet et al.,

2003) and is thought to have an important physiological role in cell volume regulation in

cardiomyocytes (Calloe et al., 2007), mammary epithelial tissues (vanTol et al., 2007),

liver cells (Lan et al., 2006)... Many theories have been proposed on how the channel

activity is triggered during cell volume changes. One possibility was that cell volume

sensitivity could be mediated by membrane stretch. We have recently disproved this

hypothesis and shown that cell membrane stretch and cell volume change are two

independent mechanisms that can regulate BK and KCNQ1 channels (Hammami et al.,

2009). In several studies it was shown that hypotonic cell swelling induces ATP release

which can activate ion channels eg. Volume activated Cl- channel (Wang et al.,

1996;Roman et al., 1999;Perez-Samartin et al., 2000;Darby et al., 2003) and potassium

channels (Hafting et al., 2006). Therefore, the goal of our study was to investigate

whether ATP release is involved in volume mediated response of KCNQ1 channels. For

this purpose we expressed KCNQ1 ± AQP1 in Xenopus oocytes.

Materials and methods

Expression in Xenopus laevis oocytes

cDNAs coding for Aquaporin1 (AQP1) and KCNQ1 were subcloned into expression

vectors and expressed in Xenopus laevis oocytes. Xenopus laevis oocytes were isolated


68

and defolliculated as previously described (Grunnet et al, 2002) or purchased from

Ecocyte Bioscience (Germany). Synthetic RNA was prepared by in vitro transcription (T3

and T7 mMessage machine kit from Ambion) from DNA templates (coding for AQP1

and KCNQ1) linearized with Pst1 and XbaI (New England Biolabs, Ipswich, MA, USA)

for AQP1 and KCNQ1 respectively. RNA was extracted by MegaClear kit (Ambion). 50

nl of mRNA was injected in oocytes, which were then kept in Kulori medium (in mM:

90 NaCl, 1 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES-Tris, pH 7.4) at 19°C.

ATP release measurements

ATP released from individual defolliculated oocytes (non-injected, KCNQ1 injected and

KCNQ1+AQP1 injected) was monitored 3 days after RNA injection using luciferin-

luciferase bioluminescence assay (FLAA, Sigma-Aldrich) similar to by Maroto and

Hamill 2001 with slight modifications. Individual oocytes were placed in a 96 well plate

containing 45 μl Kulori and 5 μl of the Sigma ATP assay reagent in each well (1 mg/ml

luciferin-luciferase mix). Oocyte handling and transfer to the well will elicit an increase

in ATP release, therefore oocytes were left to rest in the well for 1 hour before ATP

release was monitored using Victor luminometer (Perkin Elmer). After this first

measurement, 22.5 μl of 50% Kulori + 2,5 μl luciferin-luciferase mix (LL-mix) or 22.5 μl

Kulori + 100 mM mannitol + 2.5 μl LL-mix was added carefully by pipetting in order to

swell or shrink the oocyte respectively (∆50 mosmol). Immediately after, release of ATP

was measured. Osmolarities of the solutions used here correspond to the same ones done

with the two electrode voltage clamp measurements (see next section). For control, 22.5

μl Kulori +2.5 μl LL-mix was added in order to subtract any pipetting effect. For both

standards and oocyte experiments the ATP induced light was measured over a 60 sec

sampling period. The background signal (a blank) was measured and subtracted from

samples. Standard curves were performed by plotting the log of luminescence intensity


69

(relative luminescence units) against the log of ATP concentrations (moles/liters) with the

different osmolarities using the Sigma ATP calibration standards. The ATP induced light

was converted to moles/L of ATP concentration according to the standard curves

prepared each day.

Electrophysiological measurements

All measurements were performed 3 days after RNA injection using a conventional two-

electrode voltage-clamp set-up. The measurements were done in medium that was

isotonic (65 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 50 mM mannitol, 5 mM

Hepes, pH 7.4 (188 mosmol kg−1

)), hypotonic (65 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1

mM CaCl2, 5 mM Hepes, pH 7.4 (137 mosmol kg−1

)) or hypertonic (65 mM NaCl, 1 mM

KCl, 1 mM MgCl2, 1 mM CaCl2, 100 mM mannitol, 5 mM Hepes, pH 7.4 (239 mosmol

kg−1

)). The chemicals Apyrase and ATP from Sigma Aldrich were added at the different

conditions.

Data acquisition and analysis was performed with Clampex 10 and clampfit 10

(Molecular devices) software programs, respectively. GraphPad Prism 4 was used for

preparing graphical displays.

Statistics

If nothing else mentioned, numerical data are presented as means ±SEM with n

observations in different oocytes. Comparisons are made by using Student‟s two tailed,

unpaired or paired t-test depending on the data.

Results

1) Basal ATP release at rest and during changes in cell volume


70

In order to determine whether there is ATP release during cell volume changes in

Xenopus oocytes at the different osmotic conditions (50 mosmol difference), luciferin

luciferase bioluminescence assay was applied. Figure 1A shows standard curves of

bioluminescence assay in Kulori and after adding Kulori, hypotonic and hypertonic

solution. The assay sensitivity was not reduced in the different solutions.

By monitoring ATP release just after handling and transfer of oocytes to the wells we

noticed a very high ATP release due to the mechanical perturbations, which decreased

with time (not shown). We therefore let the oocyte rest for one hour after they were

transferred to the wells.

Basal ATP release was measured 1 hour after oocyte transfer to a well containing Kulori

and luciferin luciferease mix. Figure 1B shows difference in basal ATP release between

non-injected (CTRL) oocytes; AQP1, KCNQ1 and AQP1+KCNQ1 injected oocytes at

rest. Surprisingly, AQP1 injected oocytes had nearly the same basal ATP release as the

control oocytes, whereas basal ATP release for KCNQ1 injected oocytes was 3-4 fold

higher.

We predicted that oocytes expressing AQP1, which are rapidly undergoing changes in

cell volume, will release ATP to the surrounding medium at a higher rate within the first

minutes compared to controls ones. In order to test for this hypothesis, ATP release was

monitored before and after pipetting hypo - or hyperosmotic solution to each well. Since

pipetting may induce mechanically ATP release, as control pipetting effect was also

measured after adding Kulori. Pipetting had no significant effect on ATP release in non-

injected control oocytes, whereas it had an effect on the injected KCNQ1 ± AQP1 as

shown in Figure 2A.


71

Figure 2B shows that in non-injected control oocytes swelling did not induce further

release of ATP compared to oocytes subjected to Kulori in Figure 2A. In contrast, the

injected oocytes KCNQ1+AQP1 had a 36 % further release of ATP to the extracellular

medium when subjected to cell volume increase. Surprisingly, we did not expect ATP

release for the oocytes expressing only KCNQ1 since these do not volume regulate. In

another experiment, hyperosmotic exposure (Figure 3C.) and hence shrinkage also

induced ATP release for KCNQ1+AQP1 and KCNQ1. A significant ATP release was

also seen for the non-injected oocytes in response to shrinkage.

From these data we can conclude that during changes in cell volume there is indeed an

ATP release, but this is independent of AQP1. The question whether this increase in ATP

release is coupled to the volume sensitivity of KCNQ1 channels is addressed in the

following section.

2) Effect of apyrase on KCNQ1 currents

To investigate if the response of KCNQ1 to changes in cell volume involves the

purinergic signaling pathway, we added the ATP/ADP hydrolyzing enzyme apyrase at the

different osmotic conditions. Figure 3 shows that application of 7U/ml apyrase decreased

the overall KCNQ1 current level. The current also decreased in increasing concentrations

of Apyrase (not shown).We tested whether simply the presence of protein may have an

effect on the current. Therefore we performed similar experiment with Bovine serum

albumin at similar concentrations (not shown). However this did not have any effect. We

propose that apyrase has an unspecific effect with the KCNQ1 channel. The decrease in

current in all conditions with apyrase may also be due to the hydrolyzing effect on the

basal ATP release close to the membrane of KCNQ1 injected oocytes.


72

After normalization of the current to isotonic condition in figure 3 B. the relative

percentage changes with hypo or hyper solution were the same with and without

application of apyrase. This indicates that the volume sensitivity of KCNQ1 was

relatively unchanged.

3) Effect of added extracellular ATP on KCNQ1 currents

In order to further test whether KCNQ1 current response to volume changes is mediated

by extracellular release of ATP, 100 μM ATP was added to the extracellular solution (Iso,

hypo and hyperosmotic solutions) and current was recorded at the end of + 40 mV

depolarizing potential. Figure 4 shows that the KCNQ1 current did not decrease or

increase in the presence of 100 μM extracellular ATP relative to control solutions. Higher

concentrations of ATP were also tried with no effect (not shown). These data indicate that

ATP is not involved in the increase in current during hypoosmotic conditions or decrease

during hyperoosmotic conditions.

Discussion

In the present study, we show that mechanical stimulation and changes in cell volume

induces ATP release for oocytes expressing KCNQ1 with and without AQP1.

Extracellular given ATP and apyrase however, has no significant effect on KCNQ1

current response to changes in cell volume.

In this study, we measured ATP release at resting conditions and during changes in cell

volume.

Basal ATP release: For non-injected control oocytes, basal release corresponded to an

equivalent of 19 ± 2.65 femtomol of ATP in 50 μl Kulori. This value is in agreement with


73

previous results where basal ATP release for non-injected oocytes was around 20

femtomol (Maroto & Hamill, 2001). Note that this is very small concentrations as we are

working in large volumes (50 μl); however this may be larger in close proximity to the

oocyte surface. Another possible explanation for the low ATP concentrations, is the

endogenous expression of the membrane protein CD39 which hydrolysis ATP and ADP

(Aleu et al., 2003).

The KCNQ1±AQP1 injected oocytes had a 4 fold higher basal ATP release than the

AQP1 injected oocytes. It seems like the type of membrane protein expressed may have

different effects on the basal ATP release. Heterologous expression of KCNQ1 channel

may have several consequences: 1) the oocyte is stressed due to a high synthetic rate of

proteins and high rate of exocytosis, which may lead to an increase in the release of ATP

present in the exocytotic vesicles; 2) KCNQ1 may be functionally interacting and

upregulating the insertion of ATP transporters to the membrane; or 3) KCNQ1 may be

permeable to ATP. In fact, several mechanisms have been suggested regarding how ATP

is released. It can be released by a non-ionic process through mechanical induced

constitutive release of vesicles (Maroto & Hamill, 2001) and through hormonal/neuronal

regulated exocytosis (Lazarowski et al., 2003). Moreover release can take place by a

conductive ionic process through membrane channels, such as mechanically gated ion

channels, hemichannels such as connexins and pannexins, maxi anion channels, volume

regulated anion channels, CFTR and P2X7 receptors (reviewed by (Corriden & Insel,

2010)). It is unlikely to consider the cation selective KCNQ1 channel as an ATP

conductive channel, since ATP is negatively charged in physiological solutions (ATP4-

).

ATP release during cell volume changes: In this study, ATP release was measured under

iso-, hypo and hyperosmotic conditions for non-injected control oocytes and KCNQ1 ±

AQP1. Hypoosmotic induced ATP release is widely reported in many native and cultured


74

cells (Wang et al., 1996;Shinozuka et al., 2001;Boudreault & Grygorczyk, 2004;van der

et al., 1999). However, previous studies where defolliculated, non-injected Xenopus

oocytes were tested in hypotonic stress conditions (Δ140 mosmol/l) did not detect any

release of ATP (Aleu et al., 2003;Maroto & Hamill, 2001). In contrast oocytes challenged

with hypertonic solution (Δ300 mosmol/l) released ATP (Aleu et al., 2003). This

behavior is also what we see for our control non-injected oocytes (cf. Figure 2 B and C).

In contrast to control oocytes we detect ATP release in the injected oocytes

(KCNQ1±AQP1) both during hypotonic and hypertonic conditions. We may assume that

when expressing an exogenous channel, the exocytotic pathway is boosted and in parallel

swelling the oocyte, may intensify the response thereby contributing to a higher release of

vesicles containing both the channel proteins ready to be inserted to the membrane and

the ATP molecules ready to be released to the outside. This explains the higher ATP and

the increase in KCNQ1 current detected during volume increase.

During volume changes, we would expect that the AQP1+KCNQ1 expressing oocytes to

be most volume sensitive and therefore we would expect to see a higher ATP release.

However we see a similar increase in ATP release in the KCNQ1 injected oocytes. This

indicates that, larger volume changes in the presence of AQP1 do not induce a higher

release of ATP and that the rate of release is independent on AQP1.

The released ATP in Xenopus oocytes during cell shrinkage was previously shown to be

coupled to the activation of an inward current (Zhang & Hamill, 2000;Aleu et al., 2003).

There could be a possibility that different ATP release mechanisms operate under

hypoosomotic and hyperosmotic in the injected oocytes through an exocytotic pathway

during cell swelling and through a conductive channel during cell shrinkage.

Exogenous ATP and apyrase


75

In another series of experiments ATP and Apyrase were added to the extracellular

medium in order to investigate whether the response of KCNQ1 to changes in cell volume

involves the purinergic signaling pathway. Neither ATP nor apyrase had any effect on the

volume induced changes in KCNQ1 currents. A possible explanation for the absence of

any effect could be that either ATP and apyrase does not reach the micro-environments of

the channels (Joseph et al., 2003) or because of the extracellular ATP degradation by

CD39.

ATP exerts its function by activating purinergic receptors at the cell membrane that

subsequently modulates ion channels through intracellular signaling pathways involving

an increase in intracellular calcium concentration. However, the existence of endogenous

purinergic receptors in Xenopus oocytes is puzzling. Many studies based on

electrophysiological experiments have shown that follicular cell-enclosed oocytes are

endowed with purinergic receptors that respond to ATP evoking a current response

(Lotan et al., 1986;Arellano et al., 1996;Arellano et al., 1998;Arellano et al., 2009;King

et al., 1996a;King et al., 1996b) whereas defolliculated oocytes are devoid of purinergic

receptors according to the fact that ATP failed to activate any current response. However

these assumptions are based on studies done a few hours after defolliculation of oocytes

and therefore the receptors could be desensitized or internalized. One single study

however has shown the expression of endogenous Xenopus oocyte adenosine receptor at

the membrane (Kobayashi et al., 2002). To our knowledge, there are no published data on

purinergic receptor mRNA expression nor receptor protein expression on defolliculated

oocytes. Moreover by using intracellular calcium changes as a functional indication of the

presence of purinergic receptors seems to be problematic since Ca2+

signals are difficult

to detect during volume changes in Xenopus oocytes (Vandorpe et al., 1998;Grunnet et

al., 2002;Locovei et al., 2006)


76

Whether purinergic receptors are present or not is unclear, but ATP seems not to induce

KCNQ1 current or volume sensitivity of the channel. It is unlikely that purinergic

signaling is involved in KCNQ1 volume response in the Xenopus oocytes. A

disadvantage in using Xenopus oocyte as an expression system is its amphibian origin and

“egg status” which may lack some components that normally present in warm blooded

animal cells. Interestingly, previous studies have shown a coupling of the KCNQ1 to

purinergic signaling in native tissues. The strial marginal cells and vestibular dark cell of

the inner ear of rodents expresses KCNQ1, its auxiliary β-subunit KCNE1 and purinergic

receptors (P2Y4) at the apical membrane which have been shown to have an important

function in endolymph homeostasis and protection from overstimulation (Housley et al.,

2009;Lee & Marcus, 2008). KCNQ1/KCNE1 channel activity and thus K+ secretion was

shown to be modulated by 3 purinergic pathways in rodents when P2Y4 receptors are

stimulated: 1) G protein-PLC activation leads to consumption of membrane PIP2 with the

consequent reduction of K+ channel activity 2) the DAG-PKC path decreases K

+ channel

activity directly via phosphorylation of the channel and 3) the IP3/Ca2+ path decreases

the channel activity directly via the effect of Ca2+ on channel activity (Lee & Marcus,

2008).

Additionally, Honoré et al (1992) have reported that stimulation of purinergic receptors

regulates the KCNQ1/KCNE1 channel activity in mouse heart. This study was concluded

by injection of cardiac polyA+ RNA from neonatal mouse heart into Xenopus oocytes.

The RNA directed the expression of IsK channel as well the expression of purinergic P2

receptors. By stimulating these receptors it produced intracellular Ca2+ increase, DAG

and thereby activating protein kinase C which increased IsK activity (Honore et al.,

1992).


77

These studies indicate that there is indeed a coupling of KCNQ1/KCNE1 channels with

purinergic signaling. In our study where only KCNQ1 is expressed, KCNE1 is maybe

necessary in order to elucidate whether purinergic signaling has an effect on KCNQ1

volume response.

In conclusion, we showed that oocytes release ATP in response to mechanical, hypo and

hypertonic loads which may be due to different mechanisms. Also expression of KCNQ1

involves ATP release but AQP1 does not. Furthermore extracellular added ATP and

apyrase had no effect on volume induced currents. These results indicated that purinergic

signaling is not involved in volume mediated KCNQ1 activity in our expression system.

However we cannot exclude that other components e.g. KCNE1 could do the job.

Acknowledgements: The authors thank Ms. Z. Rasmussen for expert technical

assistance. This work has been supported by FNU and Carlsberg Foundation (oocyte

setup).


78

Figure legends

Figure 1 A) Standard curves of the bioluminescence assay in Kulori, hypotonic and

hypertonic solution. B) Basal ATP release difference from non RNA injected oocytes

(CTRL n= 116) and injected oocytes (AQP1 n=31, KCNQ1 n=97 and KCNQ1+AQP1

n=115) at resting condition. The data represent mean±SEM. Oocytes from 7 different

frogs. *** P < 0.001; NS: non significant.

Figure 2 ATP release before and after exposing oocytes to (A) Kulori, (B) hyposmotic

and (C) hyperosmotic solutions. In (A), pipetting had a slight increasing effect on ATP

release in Q1 and Q1+AQP1 injected oocytes. In (B), hypososmolar solution induced a

higher ATP release in the injected oocytes independent of whether AQP1 was present or

not. In (C), hyperosmolar solutions had similar effect on ATP release in all cases. The

data represent the mean ± S.E.M. (oocytes from 7 different frogs). (Kulori n= 35-40,

Hypo n= 36-44 and Hyper n= 26-34) NS: non significant, * P < 0.05, ** P < 0.01 and ***

P < 0.001.

Figure 3 Effect of application of extracellular apyrase on KCNQ1 current response to cell

volume changes. A. Original KCNQ1+AQP1 current traces at 40 mV depolarizing

potential during osmotic challenges with and without the presence of apyrase. B.

Columns show the changes of current at the end of a depolarization period (+40 mV)

during osmotic challenges before (CTRL) and after addition of apyrase. The data

represent the mean± S.E.M. n=5 oocytes.

Figure 4 Effect of extracellular ATP on KCNQ1 current response to cell volume changes

(coexpressed with AQP1). A. Original KCNQ1+AQP1 current traces at 40 mV

depolarizing potential during osmotic challenges with and without the presence of 100

μM ATP. B. Columns show the changes of current at the end of a depolarization period


79

(+40 mV) during osmotic challenges before (CTRL) and after addition of 100 μM ATP.

The data represent mean± S.E.M. n=6 oocytes.


80

Figure 1

Figure 2


81

Figure 3

Figure 4


82

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86

Manuscript III: KCNE1-induced increase in KCNQ1 currents is not

mediated through enhanced plasma membrane expression

Sofia Hammami1,2

, Dan A. Klaerke2 & Niels J. Willumsen

1

1Department of Biology, Faculty of Science, University of Copenhagen, Denmark;

2Department of Physiology and Biochemistry, IBHV, Faculty of Life Sciences, University of

Copenhagen, Denmark

Corresponding author:

Niels J. Willumsen

[email protected]

Telephone: +45 35321635 and fax number: +45 3532 1567

University of Copenhagen

Department of Biology

The August Krogh Building

Universitetsparken 13

2100-Copenhagen Ø

Denmark

mailto:[email protected]


87

Abstract

Association of the voltage activated potassium channel KCNQ1 with the accessory

protein subunit KCNE1 gives rise to the cardiac IKs delayed rectifier potassium current.

Aside from altering the kinetic characteristics of the KCNQ1 channel current, KCNE1

also augments the KCNQ1 current. However it is debated whether this increase in

macroscopic current is due to an increase in ion channel conductance (γ), the open state

probability (Po) or an increase in the number of channels in the plasma membrane (N).

The latter can be quantified by measuring the level of KCNQ1 surface expression by

using an enzyme-linked immunoassay. To do this, we employed a HA-tagged version of

the KCNQ1 channel and expressed it with and without KCNE1 in Xenopus oocytes. The

HA-tag, which is located at the extracellular side of the protein, allowed us to “count” the

number of KCNQ1 channels expressed in the cell membrane. In parallel, currents were

measured with two electrode voltage clamp. The results show that the KCNQ1 surface

expression is lower when KCNE1 is coexpressed compared to KCNQ1 alone despite the

higher current for the heteromeric KCNQ1/KCNE1. This indicates that the overall

increase of the KCNQ1 current when KCNE1 is coexpressed is not due to an increase in

ion channel surface density but rather to an increase in single-channel conductance or in

open state probability.

Keywords

Surface expression, KCNQ1, KCNE1, IKs current, Xenopus oocytes


88

Introduction

KCNQ1 potassium channels are abundant in the cell membranes of cardiac muscle where

they are involved in repolarisation of the cardiomyocyte membrane after an action potential.

In addition, KCNQ1 channels have been found in a number of epithelial tissues and have

been demonstrated to be essential for transepithelial transport and for participating in

potassium absorption and secretion in the inner ear, the kidney, lung, stomach, and intestine

[1]. KCNQ1 channel activity is regulated by many factors such as phosphorylation [2], cell

volume [3] and regulation by β-subunits [1]. KCNQ1 associates with a number of accessory

proteins such as KCNE1[4], KCNE2 [5], KCNE3 [6], KCNE4 [7], KCNE5[8] and

corticosteroid hormone induced factor CHIF [9], which all have overlapping tissue

distribution with KCNQ1. These auxiliary proteins which are small with only one-

transmembrane-segment alter the function of the KCNQ1 by modifying its

electrophysiological properties. Heteromeric association of KCNQ1 with KCNE1 (also

denoted MinK or IsK) induces a current with electrophysiological channel properties

markedly different from that of the KCNQ1 channel itself, but similar to the cardiac delayed

rectifier potassium current (IKs) which contribute to the repolarisation following a cardiac

action potential.

When expressed alone, the KCNQ1 gives rise to a voltage dependent outward current upon

depolarization which reaches a steady state within 1s. Subsequent repolarisation elicits a tail

current with an initial increase in the amplitude (a „hook‟) before deactivation. This tail

current hook is resulting from recovery of the channels from inactivation at a rate faster than

deactivation [4]. However co-assembly of the regulatory β-subunit KCNE1 with KCNQ1

results in a significant change of the electrophysiological properties of the channel. The

voltage activation threshold is shifted to a more positive potential, the deactivation is much

slower and the inactivation is almost completely absent [4]. Moreover the current is much


89

larger than the one induced by KCNQ1 alone. This increase in macroscopic current can be

due to an increase in single channel conductance, an increase in the number of functional

channel complexes in the membrane or increase in the channel open state probability Po.

Controversies have occurred when determining which of these parameters are altered.

Romey et al. [10] concluded that the effect of minK coexpression was twofold: a decreased

single channel KCNQ1 conductance (from 7.6 to 0.6 pS) which was overruled by an

increased channel density resulting in the overall enlargement of the macroscopic current.

However studies made by Yang and Sigworth (1998) [11] and Pusch (1998) [12] led to the

opposite conclusion: the observed current increase upon coexpression with minK was

contributed to an increase in single channel conductance. In noise analysis studies, these

investigators demonstrated a larger single-channel conductance of heteromeric

(KCNQ1/KCNE1 complex; γ = 4.5 pS) compared to homomeric channels (KCNQ1 alone;

γ = 0.7 pS).

This present study focus on a comparison of the number of KCNQ1 channels in the plasma

membrane with and without the presence of the auxiliary subunit KCNE1 through enzyme

linked immunoassay on HA tagged KCNQ1 proteins. This allows us to determine if the

number of ion channels (N) in the plasma membrane is altered when KCNQ1 is coexpressed

with KCNE1.


90

Materials and methods

Expression in Xenopus laevis oocytes

Xenopus laevis oocytes were isolated and prepared as previously described[13]. cDNAs

coding for human KCNQ1, KCNQ1-HA [14] and KCNE1 were subcloned into pXOOM

vectors and expressed in Xenopus laevis oocytes [15]. KCNQ1-HA has two

hemagglutinin epitopes inserted in the extracellular site between the S3 and S4 segment

of the KCNQ1 protein (insert, figure 1B). Synthetic RNA was prepared by in vitro

transcription (T7 mMessage machine kit from Ambion) from DNA templates linearized

with XbaI and NheI (New England Biolabs, Ipswich, MA, USA) for KCNQ1-HA and

KCNE1 respectively. RNA was extracted by MegaClear kit (Ambion). 50 nl of mRNA

was injected in oocytes which were then kept in Kulori medium (in mM: 90 NaCl, 1 KCl,

1 MgCl2, 1 CaCl2, 5 HEPES-Tris, pH 7.4) at 19°C. For coexpression of KCNQ1(-HA)

and KCNE1 subunit, the mRNAs were mixed in equal molar ratios. We also injected the

same amount of water than KCNE1 to have the same diluted volume of KCNQ1.

Electrophysiological measurements

All measurements were performed 4 to 6 days after RNA injection using a conventional

two electrode voltage clamp setup. The measurements were done in Kulori medium.

Surface expression

Measurements of surface expressed proteins through enzyme immunoassay were

conducted right after current measurements on the same oocytes. The hemagglutinin

epitopes which are inserted in the extracellular site between the S3 and S4 segment of the

KCNQ1 protein allows us to measure the amount of surface expressed protein through

enzyme immunoassay. The procedure was conducted as previously described [16] with

slight modifications: Oocytes were placed in Kulori with 1% bovine serum albumin


91

(BSA) for 1 hour to block nonspecific binding. They were then incubated for 60 min in

0.5 µg/ml primary antibody (3F10 anti HA from Roche molecular Biochemicals) solution

prepared in Kulori-BSA(1:200), then washed with Kulori-BSA six times by moving the

oocytes from one petri dish to another changing to a new pasteur pipette at each time.

Oocytes were then incubated with the secondary antibody solution (goat anti-rat F(ab´)2

fragments (Jackson Immunosearch Laboratories) at a 1:400 dilution) also prepared in

kulori-BSA for 1 hour, then washed 4 times with kulori-BSA and 4 times without BSA.

Five oocytes were then placed in 200 µl OPD luminescence solution (OPD tablets from

sigma) in a well of a 96 well plate. They were incubated for one hour and subsequently

100 µl of the solution was moved to another plate where absorbance measurement was

conducted on the absorbance reader Victor (Perkin Elmer) at 450 nm. All steps were done

on ice and 4ºC solutions in order to minimize endocytosis. Background noise from

injected oocytes expressing non-tagged protein has been subtracted. The light emitted by

the OPD reaction was expressed as relative light units (RLU) reflecting the amount of

surface expressed proteins.


92

Results

Comparison between KCNQ1 and KCNQ1-HA

KCNQ1-HA has two HA epitopes located in the second extracellular loop, i.e. between

the third and fourth transmembrane segment of the channel protein (see insert in figure

1B). In initial experiments we examined if the HA-tags had any influence on the current

kinetics of the channel by comparing it with the original non-tagged channel KCNQ1.

The HA-tagged channel exhibited almost identical electrophysiological properties. Figure

1 shows that although overall current was lower for KCNQ1-HA than for KCNQ1, the

current showed the same kinetic behavior: an outward rectifying current which reaches a

plateau phase within ~1 sec and the characteristics tail current with the hook. For

KCNQ1 and KCNQ1-HA the half maximal V½ was -10.6 ± 0.9 mV and -8.1 ± 0.9 mV,

respectively, and the slope factor was 17.8 ± 0.9 mV and 27.9 ± 1.0 mV, respectively,

indicating similar activation behaviors (Figure 1C, upper panel). It has earlier been shown

that the HA-tags do not interfere with channel trafficking [14].

When KCNQ1-HA was coexpressed with KCNE1 it induced a slowly activating current

as seen for the non-tagged protein however with minor differences compared to KCNQ1

+ KCNE1 (Figure 1C, lower panel). Recent studies which determined the KCNE1

structure by solution NMR and subsequently experimentally restrained docking of the

KCNE1 transmembrane domain (TMD) into the KCNQ1 channel, have revealed that the

curved nature of KCNE1 enables the TMD to form extensive contacts with a cleft formed

between the upper part of S3 on the voltage sensor of one KCNQ1 subunit and the S5 and

S6 segments of another KCNQ1 subunit [17]. As the HA tags in our construct are placed

between the S3 and S4 domain (where KCNE1 actually make contact), it is likely that

they affect the interaction between KCNE1 and KCNQ1. This can possibly explain the

difference in the I/V relationship, we see when KCNQ1-HA is coexpressed with KCNE1


93

as we see no differences between KCNQ1-HA and wild type KCNQ1 when expressed

alone (figure 1C).

The steady state current was also lower for KCNQ1-HA + KCNE1 than for KCNQ1 +

KCNE1, but most importantly, we observed an almost identical effect of KCNE1 on the

fractional increase in current when coexpressed with either KCNQ1-HA or KCNQ1 (Cf.

Fig 1A + B). Thus, it seems reasonable to measure the level of KCNQ1-HA surface

expression by enzyme-linked immunoassay in order to determine whether the number of

surface expressed KCNQ1 channels is increased or decreased when coexpressed with

KCNE1 β-subunit.

Surface expression over time, a test for the sensitivity of the HA-tag system

In another series of experiments the sensitivity of the enzyme-linked immunoassay was

examined. For oocytes injected with mRNA coding for the HA-tagged KCNQ1 channels,

steady state current as well as the HA-signal were measured on the 4th

and 6th

day after

injection. As expected, current increased from day 4 to day 6 after RNA injection

indicating that an increased number of ion channels were expressed in the plasma

membrane (Fig. 2, black columns). In consistence with this, parallel experiments showed

an increased signal from exposed HA-tags (Fig. 2, scattered columns). From day 4 to day

6, the steady state current increased 44 % (from 0.93 ± 0.15 to 1.34 ± 0.16 µA) and the

HA-tag signal simultaneously increased 54% (from 0.0076 ± 0.0008 to 0.0118 ± 0.0024

RLU ). These results indicate that for the HA-tagged KCNQ1 channels, the number of

channels expressed in the plasma membrane is reflected with reasonable precision in the

signal from the the enzyme-linked immunoassay. Therefore, this assay seems to be a

relieable and sensitive method to “count” the number of channels in the plasma

membrane.


94

Surface expression versus current. KCNE1coexpression does not increase the number

of KCNQ1 ion channels translocated to the membrane surface

In order to investigate whether the current induced by KCNE1 is affecting the number of

ion channels that are translocated to the membrane, oocytes were injected with mRNA

coding for KCNQ1-HA + water or a mixture of mRNA coding for KCNQ1-HA and

KCNE1. Four days after the injection, the current and surface expression for KCNQ1-

HA+ KCNE1 and KCNQ1-HA + H2O was measured. The measurements show that, the

current amplitudes are close to equal for KCNQ1-HA + H2O (0.54 ± 0.07 µA) and

KCNQ1-HA+ KCNE1 (0.59 ± 0.11 µA), whereas the surface expression for the

homomeric channel is significantly higher (0.0048 RLU) than for the heteromeric channel

(0.0014 RLU). The experiments shown in Figure 2 indicate that the surface expression

measurements to some extent can be considered quantitative. Thus, the data in Figure 3

suggests that the membrane expression for KCNQ1-HA + KCNE1 in the present studies

is approximately 3.5-fold lower than for and KCNQ1-HA although the currents

conducted by the channels are equal. This indicates that KCNE1 is not inducing an

increased number of KCNQ1 channels in the membrane but is rather acting on the ion

channel conductance or open probability.


95

Discussion

The mechanism for the increased current seen in electrophysiological measurements

when the KCNQ1 channel is coexpressed with KCNE1 has previously been investigated

by fluctuation analysis in a number of studies [10-12]. However, the results have been

conflicting, and it is at present not clear whether the presence of KCNE1 affects the

membrane expression, the open probability or the single channel conductance.

In the present study, we address this unresolved question by employing an alternative

approach, which relies on the detection of surface-bound antibodies using enzyme-linked

immunoassay to elucidate whether the number of functional ion channels is altered after

coexpression of KCNQ1 with KCNE1. By using an enzyme-linked immunoassay and

HA-tagged version of the KCNQ1 channel we were able to estimate the number of ion

channels present in the plasma membrane in the presence and absence of KCNE1.

Our results show that co-expression of the KCNE1 subunit in fact decreases the

membrane expression of the KCNQ1 alpha-subunit, but increases the current through

each expressed channel by a factor of approximately 3.5 (Cf. results section and Fig. 3).

Our results do not allow us to determine whether this increase in current in mediated

through increased open probability, increased single channel conductance or both.

However, currents for KCNQ1 as well as KCNQ1/KCNE1 are close to saturation after

depolarisation to 60 mV for 4 s (Cf. Fig. 1B), and it could be assumed that the open state

probabilities for homomeric and heteromeric ion channels are equal and close to unity at

60 mV. If that is the case, then our results indicate that the single channel conductance for

the heteromeric KCNQ1/KCNE1 channels is approximately 3.5-fold higher than for the

homomeric KCNQ1 channels. This estimate would agree with noise analysis studies

made by Yang and Sigworth [11], Pusch [12] and Sesti and Goldstein [18] who all

estimated a 3-4 fold increase in single channel conductance upon co-assembly of KCNE1


96

and KCNQ1. In contrast, our results are not consistent with Romey et al. (1997); these

authors concluded that the interaction reduces KCNQ1 conductance from 7.6 to 0.6 pS

and at the same time dramatically increases the KCNQ1 channel density in the plasma

membrane.

Since it is well described that the presence of KCNE1 increases the whole cell KCNQ1-

current, it may seem surprising that co-expression of KCNE1 in fact decreases the

number of ion channels in the plasma membrane. However, as argued above, the

explanation may be a large increase in single channel conductance induced by the

presence of KCNE1. In the present study, the decrease in the number of channels in the

plasma membrane is quite significant, and it cannot be excluded that this is in part due to

“saturation” of the expression system. However, since our results are based on the ratio

between measured current and the presence of HA-tagged KCNQ1 in the plasma

membrane, such a phenomenon would not affect our conclusions. In addition, the effect

of co-expression of KCNE1 or KCNE2 on KCNQ1 channels was recently examined in

another expression system (COS-7 cells). Also this study showed that cell surface

expression of KCNQ1 was significantly lower as compared to expression of KCNQ1

alone [19]. It cannot be excluded that the presence of KCNE1 also in intact tissue may

decrease the membrane expression of KCNQ1 although the opposite has also been shown

to be the case [20]. This assumption is supported by the finding that, although the

interaction between KCNQ1 and KCNE1 is dynamic, the assembly of KCNQ1 and

KCNE1 takes place in the secretion pathway before the channel complex reaches the

plasma membrane [21;22]

The KCNE1 beta-subunit (also formerly called minK) may coassemble with endogenous

KCNQ1 channels from Xenopus oocytes creating a current similar to the slow delayed

rectifier IKs identified in human heart. Thus the currents that are measured in the present


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study (see. e.g. Fig 1B) could in principle be a combination of heterologeously expressed

KCNQ1-HA/KCNE1 and endogenous KCNQ1/KCNE1. Since the membrane expression

is measured by means of the HA-tagged KCNQ1, this would give an apparent decrease in

membrane expression in our experiments. However, data from our laboratories have

shown that in such experiments the current arising from endogenous KCNQ1 channels in

the Xenopus oocytes is negligible. Also in this context, as mentioned above, it should be

kept in mind that when a different expression system (COS-7 cells, which do not express

endogenous KCNQ1 channels) was used [19], a reduction in the number of ion channels

upon co-expression with KCNE1 was measured.

Several studies have focused on the interaction of K+ channel α-subunits and accessory

proteins, how they assemble and how they affect trafficking or gating. The influence of

the different β-subunits on the α-subunits surface expression does not follow a uniform

behavior. For example the Kvβ4 subunit increases the surface expression of Kv2.2

channels without affecting the kinetic properties of the channel [23]. Other reports have

shown that KCNQ1 channel currents are inhibited by KCNE4 in mammalian cells and

Xenopus oocytes without modulating the membrane expression of KCNQ1 channels.

KChIPs 1-3 were shown to increase Kv4 current by promoting channel trafficking to the

membrane. These findings indicate that β-subunits are capable of regulating ion channel

α-subunits by either affecting the number of ion channels, the ion channel conductance or

the open channel probability. The dramatic effects we see of the association of KCNE1

with KCNQ1 on gating and permeation of the potassium current has led several

investigators to conclude that KCNE1 directly modifies the conductive properties of

KCNQ1. Indeed, there is striking evidence that KCNE1 lies in close proximity to the

KCNQ1 pore and thereby influencing KCNQ1 conducting properties and pharmacology.

Studies have shown that KCNE1 directly interacts with the S5-P-S6 pore domain and sits


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in a cleft between this pore domain and adjacent voltage sensor [17;24-26]. Moreover,

some residues from the KCNE1 transmembrane domain modulates channel activation

[26;27] whereas the juxtamembrane C-terminal domain of KCNE1 is important in

preventing channel inactivation [28;29]. Truncation of the KCNE1 C-terminus as well a

point mutation (D76N) reduced IKs current but did not affect the number of surface

expressed KCNQ1 channel proteins compared to wild type KCNE1 indicating either a

change in open probability, Po, or unitary conductance [28]. These findings further

support that an interaction of KCNE1 with KCNQ1 affects the gating and conductance

rather than the number of ion channels translocated to the membrane.

The increasing understanding of the role of the auxiliary subunit KCNE1 and its impact

on the pore forming subunit KCNQ1 has gathered substantial knowledge of the

malfunctioning of IKs channel under pathological conditions such as Long QT syndrome.

This will hopefully lead to the development of safer and more specific therapeutic drugs.

Acknowledgement: The authors thank Ms. Z. Rasmussen for expert technical assistance.

This work has been supported by The Danish Council for Independent Research -

Medical Sciences (FSS), The Danish Council for Independent Research – Natural

Sciences (FNU), The Novo Nordic Foundation, The Carlsberg Foundation, Fonden til

Laegevidenskabens Fremme and The Fouger-Hartmann Foundation.


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Figure legends

Fig. 8. Expression of KCNQ1 or KCNQ1-HA with and without KCNE1 in Xenopus oocytes.

Current/time (I/t) traces showing KCNQ1 (A) and the HA-tagged version of the KCNQ1 (B) with

(lower panels) and without (upper panels) KCNE1 recorded with TEVC in Xenopus oocytes.

Currents were activated by a step protocol from a holding potential of -80 mV. Steps of 20 mV (4

sec duration) were applied from -80 mV to 60 mV. Insert shows the location of the double HA-

epitope at the extracellular segment between S3 and S4 of KCNQ1. (C) Upper panel: Normalized

isochronal activation curves fitted with Boltzmann function for oocytes injected with KCNQ1 (V½

= -10.6 ± 0.9 mV; slope = 17.8 ± 0.9; n= 18) and KCNQ1-HA (V½ = -8.1 ± 0.9 mV; slope = 27.9

± 1.0; n= 16). Lower panel: KCNQ1+ KCNE1 (V½ = 8.9 ± 2.0 mV; slope = 28.1 ± 2.3; n= 17) and

KCNQ1-HA+KCNE1 (V½ = 124.9 ± 34.7 mV; slope = 55.0 ± 7.0; n= 15).

Fig. 9. Current and surface expression of HA-tagged KCNQ1.

Xenopus oocytes were injected with RNA coding for the HA-tagged KCNQ1 channel and the

resulting whole cell currents (black boxes) were measured with TEVC technique on day 4 and

day 6 after injection. Current were measured at Vm=60 mV at the end of 4 s depolarization step.

Subsequently surface expression measurements were made on the same oocytes by the enzyme-

linked immunoassay (scattered boxes). Data are mean±SEM. (N=5-10)

Fig. 10. Current and cell surface expression of HA-tagged KCNQ1 in presence and absence

of KCNE1.

Xenopus oocytes were injected with RNA coding for HA-tagged KCNQ1 channels and water (left

bars) or HA-tagged KCNQ1 channels and KCNE1 (right bars). Four days after injection whole

cell currents were measured (black bars) and subsequently surface expression measurements

(scattered bars) were made on the same oocytes as described above. Data are from 5 independent

experiments with 4-13 oocytes for each measurement. Data are mean±SEM. * P < 0.05, NS: Non

significant.


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Figure 1

Figure 2


101

Figure 3


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[8] K. Angelo, T. Jespersen, M. Grunnet, M.S. Nielsen, D.A. Klaerke, S.P. Olesen,

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[13] M. Grunnet, N. MacAulay, N.K. Jorgensen, S. Jensen, S.P. Olesen, D.A. Klaerke,

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Related paper: Cell swelling and membrane stretch – A common trigger of

potassium channel activation?

Ion channels are sensitive to mechanical stimuli such as cell volume changes and

membrane stretch but whether ion channel activation upon cell swelling is a result

from membrane stretch is controversial.

Mechanosensitive ion channels play important roles in many basic cellular functions. For

example, changes in volume and shape during growth division and migration subject cells

to compression, shearing and stretch. In addition, cells are constantly exposed to

mechanical stimuli from their external environment including osmotic stress, touch,

sound or gravity.

Mechanical stimuli are quantified as force per area (i.e. N/m2 or pascal, Pa), and the

applied force is detected either by membrane-bound or –associated sensor molecules that

initiate intracellular signal transduction pathways. Sensor molecules may be located either

in the general plasma membrane or in membranes of specialized sensory structures (or

„antennae‟), e.g. kinocilia and primary cilia. An important heterogenous group of

membrane proteins that include a number of mechanosensitive members is ion channel

proteins, and most, if not all, major ion channel families include mechanosensitive

members. A substantial amount of knowledge has been gathered through the later years

about the nature and function of mechanosensitive ion channels, and about their roles in a

number of human diseases. The increasing focus on mechanosensitive ion channels is

reflected in the fact that the number of scientific publications on the subject has increased

more than 10-fold over the last three decades.

Mechanosensitive ion channels act as mechanosensors by altering their ion transport

properties in response to the magnitude of the applied force. These properties include the

number of ion channels present in the membrane (N), open-state probability (Po), and

single-channel conductance (γ). Ion channel activation constitutes either a direct link that

transduces mechanical force into electrical or chemical intracellular signals, or they are

activated secondary to mechanical stimulation of other membrane-associated proteins.

Animal cells do not possess a rigid/stiff wall which can resist turgor pressure as in the

case of plant cells. Nevertheless, animal cells are able to withstand exposure to highly

hypoosmotic conditions that would result in a high hydrostatic pressure in a noncompliant


106

cell. Generally cells have excess membrane in the form of membrane invaginations and

microvilli or they may reduce their surface-to-volume ratio by attaining a more spherical

shape. During volume expansion cells increase their surface area either 1) by recruiting

excess membrane from membrane folds and smoothing out surface membrane projections

or 2) by exocytotic insertion of membrane from intracellular pools or 3) by stretching of

the membrane.

The controversy: related or independent mechanisms?

Implicitly it has been assumed that cell swelling is associated with parallel changes in

membrane stretch or tension, and that membrane stretch is the factor that elicits activation

of volume sensitive ion channels. According to this view, volume increase and membrane

stretch constitute a common mechanism in the regulation of ion channels, and,

consequently, cell swelling experiments have frequently been used as surrogate model for

membrane stretch. Experimentally, cells may be swollen osmotically in which case the

entire membrane is affected or a membrane patch may be exposed to stretch by

application of suction to a patch pipette in which case the effect is highly local.

Characteristics of membrane stretch and cell volume changes are presented in figure 1

and 2, respectively, as well as examples of their effects on ion channels.

We have aimed to clarify whether regulation of K+ channels by small changes in cell

volume and by membrane stretch indeed represents a common mechanism or whether

they can be considered independent regulatory mechanisms. This question was addressed

by taking a simple approach (Hammami et al., 2009): we studied the mechanosensitive

properties of two types of potassium channels with patch clamp technique, one which is

known to be regulated by cell volume (the KCNQ1 channel) and another, which is known

to be regulated by membrane stretch (the BK channel). The strategy was simply to test the

volume-sensitive KCNQ1 channel for stretch sensitivity, and the stretch-activated BK

channel for volume sensitivity. It was demonstrated that KCNQ1 activation in response to

cell swelling is not caused by membrane stretch, and that BK stretch-activation is not

mimicked by cell swelling (figure 3). These findings demonstrate that cell swelling is not

necessarily associated with membrane stretch, and, consequently, the two mechanisms

represent independent ways of activating mechanosensitive ion channels.

In our experiments, Xenopus oocyte volumes were varied within a physiological range, ±

approximately 5 % (27% decrease in osmolarity). Groulx (Groulx et al., 2006) has shown


107

that during moderate swelling (50% decrease in osmolarity) cells (human bronchial

epithelial cells, CHO cells and human lung carcinoma cells) increase their surface area by

30% mainly by unfolding the surface membrane. Only under extreme hypotonic swelling

(98% decrease in osmolarity) exocytotic insertion of membrane from the intracellular

pool and membrane stretch occurs. However, whether non stretch-activated ion channels

react upon this extreme membrane stretching is a question. In fact, the use of

unphysiologically large hypoosmotic shock may optimize the chance of seeing changes in

ion channel activity as a last line of defense against excessive cell swelling. Recently

Spagnoli et al. (Spagnoli et al., 2008) have shown that osmotic stress is not confined to

the cell surface /cell membrane but is distributed throughout the cell. By using atomic

force microscopy it was shown that the cell membrane did not stiffen upon cell swelling

but it became softer which was explained by the role of the cytoskeleton as a dynamic

lattice with gel-like properties.

The controversy of whether stretch- and volume-activation represent a common

mechanism is relevant to all mechanosensitive ion channels. Our data indicated that the

two mechanisms are indeed independent regulatory mechanisms. However, some ion

channels e.g. the two pore family of K+ channels TRAAK and TREK are intriguing, since

they are apparently sensitive to small changes in cell volume as well as membrane stretch

(Patel et al., 2001).

In some ion channels, stretch- and volume-sensitivity has been identified to rely on

certain amino acids in the channel-forming protein. For example, it seems well

documented that stretch sensitivity of the TREK channels is dependent on a single

positively charged amino acid located close to the cell membrane at the inner part of

transmembrane segment 2 (Honore et al., 2002). Likewise deletion of the cytoplasmic c-

terminus, the so called Stress-axis Regulated Exon (STREX), of BK channels cloned

from chick hearts, abolishes stretch-sensitivity of the channel (Qi et al., 2005). This

finding suggests that STREX constitutes a part of the mechano-sensing apparatus of the

channel.

Consequently, future studies based on site-directed mutagenesis and construction of

chimeras can likely provide insight in the roles of specific amino acids and constitute a

key to the understanding of regulatory properties of mechanosensitive ion channels. Also


108

these studies may finally establish whether the two mechanisms can be considered

independent or interrelated.

Figures

Figure 1. Membrane stretch. A. Mechanical impact such as stretch or physical bending

of the membrane leads to changes in membrane tension, thickness and curvature. These

processes are characteristic of compliant epithelia like those of the lining of the

gastrointestinal tract, airways, bladder, the endothelia, and of non-epithelial cell

membranes like muscle cells. Also, cell types experiencing wounding such as epidermal

cells, fibroblasts and migrating cells are subject to membrane stretch. B. Single channel

traces of a stretch activated channel from frog skin gland at increasing negative pressure

in the patch pipette. Three channels are present in the patch. Numbers indicate number of

open channels. C denotes the closed state. The open state probability increases with

increasing pressure (unpublished data). C. The sensitivity of ion channels to membrane

stretch can be investigated in patch clamp experiments by application of negative pressure

(suction) to a membrane patch.


109

Figure 2. Cell volume change A. Changes in cell volume result from an imbalance

between the intracellular and extracellular concentration of osmolytes that causes gain

(swelling) or loss (shrinkage) of cellular water. Subsequently the cell regulates its volume

back to the initial value by regulatory volume increase (RVI) or decrease (RVD).

Changes in cell volume occur at different physiological processes such as secretion, cell

migration, cell growth, hormone and transmitter release, synthesis or breakdown of

macromolecules. B. Time course of potassium current in Xenopus oocytes expressing

KCNQ1 and AQP1 following changes in extracellular osmolarity. The current increases

in parallel with cell swelling and decreases with cell shrinkage (Grunnet et al., 2003). C.

The volume sensitivity of ion channels can be studied in patch clamp experiments by

exposing the cell to different extracellular osmolarities thus inducing cell swelling or

shrinkage.


110

Figure 3. Membrane stretch versus cell swelling. When exposing KCNQ1 expressing

oocytes to extracellular hypoosmotic solution (cell swelling), current increases. Stretching

the membrane by applying negative pressure in the patch pipette, does not affect the

current. In contrast, cell volume changes have no effect on BK currents which are highly

sensitive to membrane stretch. The results imply that cell swelling and membrane stretch

constitute two independent ion channel regulatory mechanisms.


111

Reference List

1. Groulx N, Boudreault F, Orlov SN, & Grygorczyk R (2006). Membrane reserves

and hypotonic cell swelling. J Membr Biol 214, 43-56.

2. Grunnet M, Jespersen T, MacAulay N, Jorgensen NK, Schmitt N, Pongs O, Olesen

SP, & Klaerke DA (2003). KCNQ1 channels sense small changes in cell volume. J

Physiol 549, 419-427.

3. Hammami S, Willumsen NJ, Olsen HL, Morera FJ, Latorre R, & Klaerke DA

(2009). Cell volume and membrane stretch independently control K+ channel

activity. J Physiol 587, 2225-2231.

4. Honore E, Maingret F, Lazdunski M, & Patel AJ (2002). An intracellular proton

sensor commands lipid- and mechano-gating of the K(+) channel TREK-1. EMBO J

21, 2968-2976.

5. Patel AJ, Lazdunski M, & Honore E (2001). Lipid and mechano-gated 2P domain

K(+) channels. Curr Opin Cell Biol 13, 422-428.

6. Qi Z, Chi S, Su X, Naruse K, & Sokabe M (2005). Activation of a mechanosensitive

BK channel by membrane stress created with amphipaths. Mol Membr Biol 22, 519-

527.

7. Spagnoli C, Beyder A, Besch S, & Sachs F (2008). Atomic force microscopy

analysis of cell volume regulation. Phys Rev E Stat Nonlin Soft Matter Phys 78,

031916.


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Documents

Mechanisms underlying KCNQ1channel cell volume sensitivity Hammami.pdfThis thesis starts with a general introduction to ion channels followed by an overview of mechanosensitive ion