POLYCYSTIC RENAL DISEASE

1 in 500 autopsies

1 in 3000 hospital admissions

Accounts for ≈10% of end-stage renal failure

Autosomal dominant inheritance

CYSTIC FIBROSIS

1/2000 births in white Americans

Median age for survival late 30s

Autosomal recessive inheritance

HYDROPHOBICMOLECULES

SMALLUNCHARGED

POLARMOLECULES

LARGEUNCHARGED

POLARMOLECULES

IONS

OCONbenzene

2

2

2

H Oureaglycerol

2

glucosesucrose

H , NaHCO¯ , KCa , Cl¯Mg

+

3

2+

2+

+

+

syntheticlipid

bilayer

THE RELATIVE PERMEABILITY OF A SYNTHETIC LIPID BILAYER TO DIFFERENT MOLECULES

Intracellular Extracellular Concentration ConcentrationComponent (mM) (mM)

Cations Na 5-15 145 K 140 5 Mg 0.5 1-2 Ca 10-4 1-2 H 8 x 10-5 (pH 7.1) 4 x 10-5 (pH 7.4)

Anions Cl 5-15 110 Because the cell is electrically neutral the large deficit in intracellular anions reflects the fact that most cellular constituents are negatively charged. The concentrations for Mg and Ca are given for free ions.

COMPARISON OF ION CONCENTRATIONS INSIDE AND OUTSIDE A TYPICAL MAMMALIAN CELL

THE PROBLEM:A CELL

-

+

-

+

-

+ -

+

-

+

-

+-

+-

+

-

+

-

+

-+

- +

-

+

-

+

-

+

-

+

-

+

H O2

SOLUTIONS:

-+

+

-

+

-

+

-

+

-

+

-

-

+

H O2

-

+

+

-

+

-

+

-

+

-

+

-

-

+

- +

-

+

H O2

-

+

+

-

+

-

+

-

+

-

+

-

-

+

+

+

-

H O2

-

+

+

-

+

-

+

-

+

-

+

-

-

+

+

-

- +

-

+

-+

-+

- +

IONS

A Cross between Human Beings and Plants . . .

SCIENTISTS ON VERGE OF CREATING PLANT PEOPLE . . .

Bizarre Creatures Could do Anything You Want

Tuesday, July 1, 1980

Normal Died Asymptomatic Lethargic500

450

350

300139 baseline 119 in 2 h 122 in 3.5 d 99 in 16 d

Plasma Na concentration (mEq/l)

400

Bra

in w

ate

r (g

/10

0 g

dry

wt)

Woman drinks so much water she dies January 13, 2007

SACRAMENTO, California (AP) -- A woman who competed in a radio station's contest to see how much water she could drink without going to the bathroom died of water intoxication, the coroner's office said Saturday.

Jennifer Strange, 28, was found dead Friday in her suburba n Rancho Cordova home hours after taking part in the "Hold Your Wee for a Wii" contest in which KDND 107.9 promise d a Nintendo Wii video game system for the winner.

"She said to one of our supervisors that she was on her way home and her head was hurting her real bad," said Laura Rios, one of Strange's co-workers at Radiological Associates of Sacramento. "She was crying, and that was the last that anyone had heard from her."

Copyright 2007 The Associated Press. All rights reserved.This material may not be published, broadc ast, rewritten, or redistributed.

Simple DiffusionF

lux

[S]o

• Flux is proportional to external concentration

• Flux never saturates

PROTEIN MEDIATED MEMBRANE TRANSPORT

• PRIMARY ACTIVE

• SECONDARY ACTIVE TRANSPORT

• FACILITATED DIFFUSION

• ENDOCYTOSIS/TRANSCYTOSIS

Membrane Flux (moles of solute/sec)

• Simple Diffusion

• Carrier Mediated Transport• Facilitated Diffusion• Primary Active Transport• Secondary Active Transport

• Ion Channels

.

electrochemicalgradient

simplediffusion

channel-mediateddiffusion

carrier-mediateddiffusion

PASSIVE TRANSPORT(FACILITATED DIFFUSION)

ACTIVE TRANSPORT

transported molecule

lipidbilayer

TRANSPORT OF MOLECULES THROUGH MEMBRANES

CARRIER MEDIATED TRANSPORT

COUPLED TRANSPORT

UNIPORT

lipidbilayer

SYMPORT ORCOTRANSPORT

ANTIPORT ORCOUNTERTRANSPORT

Membrane Potential Review• The lipid bilayer is impermeable to ions and acts like an

electrical capacitor.

• Cells express ion channels, as well as pumps and exchangers, to equalize internal and external osmolarity.

• Cells are permeable to K and Cl but nearly impermeable to Na.

• Ions that are permeable will flow toward electrochemical equilibrium as given by the Nernst Equation.

Eion = (60 mV / z) * log ([ion]out / [ion]in) @ 30°C

• The Goldman-Hodgkin-Katz equation is used to calculate the steady-state resting potential in cells with significant relative permeability to sodium.

⎟⎟⎠

⎞⎜⎜⎝

⎛

∗+∗+∗∗+∗+∗

∗=outClinNainK

inCloutNaoutKm [Cl]P[Na]P[K]P

[Cl]P[Na]P[K]Plog60mVV

• Higher flux than predicted by solute permeability

• Flux saturates• Binding is selective

(D- versus L-forms)• Competition• Kinetics:

[S]o << KmM [S]

[S]o = Km M = Mmax / 2

[S]o >> Km M = Mmax

Carrier-Mediated Transport

[S]oKm

Mmax

0.5Flu

x

MEMBRANE ION TRANSPORT PROTEINS

inout

3Na

2K

ATP

ADP + Pi

Na,K-ATPase

2 major subunits

Mr=112kDß Mr≈60kD

,inhibited by ouabain,digoxin digitalis

, -H K ATPase

2subunitsMr=114kDß Mr≈60-85kD

inhibited by Schering28080, ,omeprazolelansoprazole

inout

H

K

ATP

ADP+P i

inout

2Ca

ATP

ADP+P i

-Ca ATPase

Mr=110kD

- SR Ca ATPase inhibited ,by thapsigargin

cyclopiazonic acid

- :Other P Type Ion Transport ATPases

-Cu ATPase-H ATPase-Cd ATPase

-K ATPase

1- :FoF Type ATPases

-H ATPase-K ATPase

EXCHANGERS/COUNTERTRANPORTERS:

Na/H Exchanger

Mr = 90 kD

inhibited by amiloride

inout

H

Na

Na/Ca Exchanger

Mr = 108 kD

inhibited by dichloro-benzamil, exchangerinhibitory peptide (XIP)

inout

Ca

3Na

Anion ExchangerCl/HCO3 Exchanger

Mr ≈ 102 kD

inhibited by DIDS, SITSphenyl isothiocyanate

inout

HCO3

Cl

COTRANSPORTERS:

Na,K,2Cl Cotransporter

Mr = 120 kD

inhibited by bumetanidefurosemide

Na,glucoseCotransporter

Mr = 73 kD

inhibited byphlorizin

inout

2Cl

Na,K

inout

glucose

2Na

dSCo/dt = k+ [S]o [C]o – k- [SC]o = 0 at equilibrium

k+ [S]o [C]o = k- [SC]o

k- / k+ = ([S]o [C]o)/[SC]o = Km [SC]o = ([S]o [C]o)/Km

Fractional Rate = M / Mmax = [SC]o / ([C]o + [SC]o)

M = Mmax / (1 + [C]o/[SC]o) = Mmax / (1 + Km/[S]o)

Transport Kinetics

So + Co SCo Si S = Solute C = Carrierk +

k -

Reversible Transport

Co Ci

So Si

SCo SCi

Mnet = Min – Mout =

Mmax 1 11 + Km / [S]o 1 + Km / [S]i

-( )

• Uses bidirectional, symmetric carrier proteins

• Flux is always in the directions you expect for simple diffusion

• Binding is equivalent on each side of the membrane

Facilitated Diffusion

Facilitated Diffusion: Band 3/AE1

Facilitated Diffusion: Band 3/AE1

Cytoskeletal/AE1 Interactions

Primary Active Transport: Driven by ATP

• Class P – all have a phosphorylated intermediate• Na,K-ATPase• Ca-ATPase• H,K-ATPase• Cu-ATPase

• Class V • H+ transport for intracellular organelles

• Class F• Synthesize ATP in mitochondria

Primary Active Transport: Na,K-ATPase

• 3 Na outward / 2 K inward / 1 ATP• Km values: Nain = 20 mM Kout = 2 mM• Inhibited by digitalis and ouabain• Palytoxin “opens” ion channel• 2 subunits, beta and alpha (the pump)• Two major conformations E1 & E2• Turnover = 300 Na+ / sec / pump site @ 37 °C

3 Na

2 K

ATP

ADP + Pi

Intracellular2K 3Na

ADP

E2.ATP.K2

E2.(K2)ATP

E2.(K2)

E2P.K2

3Na

E1P

E2.ATP

E1P.Na3

E1P.(Na3)

E2P.ADP.Na3E1.ATP.Na3

Pi

Extracellular2K

ATP

E2P

Na,K-ATPase Reaction Scheme

Membrane Transport and Cellular Functions that Depend on the Na,K-ATPase

COOH

SE

L KP

TYQDRVAPPGLTQIP

Q I Q K T E I S F R P N D P K S Y E A Y V L N I I R F L E K Y K D S AQ

K P C I I I K L N R V L G F K P K P P K N E S L E T Y P L T M KY

N

PNVLPVQCTGKRDEDKDKVGNIEYFGMGGFFY

T L D T E I R I E C K A Y G E N I G Y S E K D R F Q G R F D V K I E V K S

E G

G

N L

PLQYYPYYGK

K

NDESY

G Y K

L

250

LQP

Y L Q P L L A V Q F T

K

YGF

100

200

150

300

Y 50

NH2G

R

DK

Y

EPA

A

V

SE

HG

DK

K

K

S

AK

K

ER

DMD

E

LKK

E

VS

MD

DH

KL S L D

EL

HR

K

GTD

L

S

RGL

TP

A

RP

AE

IL

ARD

G

PQ

RL

T

PP

PT

TP

EW

V

K

FC

R

TNC

V

E

GT

ARG

IV

VY

T

G

DR

TV

M

GR

I

AT

LA

S

GLE

GG

QT

P

IAE

E

I

E

H

F

T

F

EPQ

T

RS

PD

F

T

NE

NP

LE

TR

N

IA

F

S

A

L H C F G

R

FI

D

E D P L LL N D V P F N V E D T D F Q F G E P F QD

I M S I L G V F C

K C R S

P

P

RA A V P A V A G I K V I M V T G D H P I T A A I G V G I I S

LY T

N

E G

YH

LR

SE E T M D K A D R P N V Q N V P I N L R A A I D

TE

N

T E I V F A R T S P Q Q K L I V E G C700

Q R

D

L

K

I

A

D D L L D S G H V V C A K

I D A K K L A P S D N V G D G T V A V IA

GQ

DF750

AT IV SV G

D V S K Q

GM A V G

IV G S A A D

N

M IL

T

S

G

EE

L

EPINS

COOH

L C

IGL YA

F

G

I

CT

T

D

LT V 800

L

L

I

D

TA

SR

E PP

E

DN

D

T

R 1000

F

L

LI I 950

Extracellular

Cytoplasmic

K

D

QL F G

G F S ML WL

I G A I

Y L G

V V L S

A V V

I I T G

C FS

YYQ

EA

KS

S

KI

M

E

S

FKN

M

VPQ

Q

A

L

VI

R

N

GE

KM

S

INA

E

D

V

VV

GD

L

VE

V KG

GD

RI

P

A

DL

R

II

S

AN

GC

KV

D

NS

S

L

G E S

I H LI T G

V A V FL G V

S F F IL S L

I L

EY T W L

E

A V I FI G I

I V A NL

V P E G

L L A T

V T V C

L T L TA

KR

MA

RK

N

N

CL

V

K

N

LEA

VE

TLG

STS

TI

CS

DK

TG

T

LT

E

QNR

M

T

VA

H

M

W

FD

N

QI

H E A D T T E Q S G V S F D K T S A T W F A L S R I A G L C N R A V F Q A N Q E N L P I L K R A VAGD

ASESLLKCIEVCCGSVMEMREKYTKIVEIPFNSTNKYQLSIHKNPNA

S

EP

K H L L V M K G A P E R I L D R C S S I L L H G K E Q P L D E E L K D A F Q N A Y L E L GGL

GERVL

G600

K A K

VE

650I

I

L

I

I

GL P L

P

AN

FI I

I

P F

M V P

A I S

L A Y

A

E

ESDI

MK

RQ

P RN

PK

TD

KLVN

ER

G 850

L I S MA Y

Q I G MI Q A

L G G FF T Y

F V IL

AENGFLP

FH

LL

GI R E T W D D R W I N D V E D S Y

GQQ 900

WT

YEQRKIVE

G LTE A

L A

F T C

H T A F

F V S

I V V V

Q W A

D L VI

CKTRRN

SV F

QQGMKN

K

FE

AF L S Y

C P G MG A A

L R MY

P L K

P W

W

TF C

A F P

Y S L

L I F

V D

EV

KL

II

RR

R

PG

G

W

V

EK

ETY

Y

100

150

200

250

300

350

400

450

500

550

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10

L ILF

Y V I F Y

GCLAGI F

M VI

TGI

LL

Q

T I

F

LGR

T

G

GSW

F

K

KDDMIFEDCGSMPSEPKERGEFNHERGERKVCRFKLDWLGNCS

MAR

G

K

AK

EE

G

SW

K

K

FI

W

N

SE

βKKE

NH2

GL

Y

52

K

Q

3/3IDENTICAL2/3IDENTICAL

NONE

4/4IDENTICAL3/4IDENTICAL

2/4IDENTICAL

NONE

1M

Amino Acid Homology Among the Na,K-ATPase Subunit Isoforms

QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.

The Na,K-ATPase As a Receptor For Signal Transduction

SR Ca-ATPase

FoF1 ATPase

Experimental Evidence for Rotation

Secondary Active Transport

• Energy stored in the Na+ gradient is used to power the transport of a variety of solutes

glucose, amino acids and other molecules are pumped in (cotransport)

Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+

(countertransport)

• These transport proteins do not hydrolyze ATP directly; but they work at the expense of the Na+ gradient which must be maintained by the Na,K-ATPase

Energy available from ATP H2OATP ADP + Pi

G = Gproducts – G reactants

Chemical Energy (G) = RT ln [C]

G = G° + 2.3 RT (log ([ADP] [Pi]) – log [ATP])

2.3 RT = 5.6 kiloJoules / mole @ 20° C

G° = -30 kiloJoules /mole @ 20°C, pH 7.0 and 1M [reactants] and [products] “Standard Conditions”

Energy Depends on Substrate Concentrations

G = -30 – 5.6 log [ATP] kJ / mole [ADP] [Pi]

The energy available per molecule of ATP depends on: [ATP] 4mM, [ADP] 400 µM, [Pi] 2 mM

per mole of ATP hydrolyzed:

G = -30 kJ – 5.6 kJ * log 4 x 10-3 2 x 10-3 * 4 x 10-4

= -30 kJ - 21 kJ = -51 kiloJoules per mole of ATP

Converting to approximately -530 meV/molecule of ATP

Energy in the Sodium Gradient

Consider Na+ movement from outside to inside:

G = Gproducts – Greactants = Ginside – Goutside

Gtotal = Gelectrical + Gchemical

Conditions for our sample calculation: Vm = -60 mV [Na+]out = 140 mM [Na+]in = 14 mM

and 2.3 RT = 60 meV / molecule

Energy in the Na Gradient: Electrical Term

Gelectrical = e mVin – e mVout

= +1e -60 mV – (+1e) 0 mV

= -60 meV

• negative sign means energy is released moving from outside to inside

• 60 meV is the energy required to move a charged ion (z=1) up a voltage gradient of 60 mV (assuming zero concentration gradient)

* *

* *

Energy in the Na Gradient: Chemical Term

Gchemical = 2.3 RT (log [Na+]in – log [Na+]out)

= 60 meV * (-1)

= -60 meV

• negative sign means energy is released moving from outside to inside

• 60 meV is the energy required to move a molecule up a 10 fold concentration gradient (true for an uncharged molecule or for a charged molecule when there is no voltage gradient)

Energy in the Na Gradient: Total

Gtotal = Gelectrical + Gchemical = -120 meV

• 120 milli-electron-Volts of energy would be required to pump a single Na+ ion out of the cell up a 10 fold concentration gradient and a 60 mV voltage gradient.

• Hydrolysis of a single ATP molecule can provide at least 500 meV of energy – enough to pump 4 Na+ ions.

• A single Na+ ion moving from outside to inside would be able to provide 120 meV of energy, which could be used to pump some other molecule, such as glucose, an amino acid, Ca2+ or H+ up a concentration gradient

Example: Na+/Ca2+ exchange

Compare the internal [Ca2+] for exchange ratios of

2 Na+ : 1 Ca2+ vs. 3 Na+ : 1 Ca2+

Vm = -60 mV, [Ca2+]out = 1.5 mM [Ca2+]in = ?

Ca2+ moves from inside to outside

G = Gproducts – Greactants = Goutside – Ginside

Gelectrical = (+2e) * (0 mV) – (+2e) * (-60 mV)

= +120 meV

Gchemical = 60 meV (log 1.5 – log ?)

Na+/Ca2+ exchange

2 Na+ 240 meV 240 = 120 + 60 log (1.5 / ?)120 / 60 = log (1.5 / ?) 102 = 1.5 / ? ? = 15 µM

3 Na+ 360 meV 360 = 120 + 60 log (1.5 / ?)240 / 60 = log (1.5 / ?) 104 = 1.5 / ? ? = 0.15 µM

Gtotal = GE + GC = 120 meV + 60 meV log (1.5 / ?)

Internal [Ca2+]can be reduced 100 fold lowerfor 3 Na : 1 Cavs 2 Na : 1 Ca

Structure of the Na/Ca Exchanger

Summary: Energetics

Transport Energetics • A molecule of ATP donates about 500

meV• It takes 60 meV to transport up a 60

mV electrical gradient• It takes 60 meV to transport up a 10

fold concentration gradient• A single sodium ion donates

approximately 120 meV

Summary: Membrane Flux (moles of solute/sec)

Simple Diffusion• Flux is directly proportional to external concentration• Flux never saturates

Carrier-Mediated Transport • Higher flux than predicted by solute permeability• Flux saturates• Binding is selective D- versus L-forms• Competition• Kinetics

Facilitated Diffusion• Uses bidirectional, symmetric carrier proteins• Flux is in the direction expected for simple diffusion• Binding is equivalent on each side of the membrane

Primary Active Transport – driven by ATP hydrolysis Secondary Active Transport – driven by ion gradients

III. Ion Channels

Transporters Regulated by Signaling Cascades

Na/H Exchangers

Na/Phosphate Cotransporter

Na/K/2Cl Cotransporter

Na/Cl Cotransporter

K/Cl Cotransporter

Na/Ca Exchanger

Na Channels

K Channels

Na,K-ATPase

H,K-ATPase

Unidirectional Transport Assays

Cells growingin multi-well plates

1. Cells washed in isotonic buffered solution

2. Required transport inhibitor(s) added

3. Flux medium containing radioactive isotope added

4. At required times flux medium rapidly removed and cells washed (3-4 x) in ice-cold isotonic saline

5. Final wash removed, cells lysed and radioactivity and protein content of samples determined

Calculations:

Specific Activity of medium:

Measure radioactivity in known volume of flux medium.

For example: For unidirectional uptake of Na into cells in medium containing:

50 mM Na100 mM choline Cl25 mM K-Hepes, pH 7.422Na (≈ 1 µCu/ml)

Measure radioactivity in 5 µl flux medium

Measure radioactivity and protein content in sample.

Determine Na influx using specific activity of mediumDetermine transport rate/protein content (Na uptake nmoles/µg protein/min)

Unidirectional Transport Assays

cpm (22Na) 1 L 1 mole cpm (22Na)

5 x 10-6 L 0.050 moles Na 109 nmoles nmoles NaX X =

THICK ASCENDING LIMB CELL

Na+

K+

Na+

K+

Na+

Lumen

Na+

K+

Na+K+

2Cl

K+ K+

Cl

Blood

HCO3

H2O

CO2

+

CA

H+

Na+

K+

H+Na+

BLOOD

"alkaline tide"

K+

H+

CO2

Cl

K+

HCO3

Cl

HCO3

Lumen

GASTRIC PARIETAL CELL

SMALL INTESTINAL CELL

Na+

K+

Na+K+

Cl 2Cl

Na+

H2O

K+cAMP

Lumen