View
8
Download
0
Category
Preview:
Citation preview
Dale Sanders
9 March 2009
Module 0220502
Membrane Biogenesis and Transport
Lecture 15
Ion Channels
Aims:By the end of the lecture you should
understand…
• The principles behind the patch clamptechnique;
• What information about ion channels canbe extracted from single channelrecordings;
• What are the main classes of ion channel inbiology, and what they do;
• How structure relates to function in theShaker class of ion channel: permeability,voltage-sensing and inactivation.
Reading• Lodish et al (2008) Molecular Cell Biology, 6th
ed. pp.458-465 & 1006-1013
More detailed, highly readable original papers, all from the lab of RodMacKinnon:
• Doyle, DA et al. (1998) The structure of the potassium channel:molecular basis of K+ conduction and selectivity. Science 405:647
• Jiang, Y. et al. (2003) X-ray structure of a voltage-dependent K+
channel. Nature 423: 33 [See also succeeding article: p. 42.]
• Zhou M. et al. (2001) Potassium channel receptor site for theinactivation gate and quaternary amine inhibitors. Nature 411:657
Relies on Giga-Ohm seal between glass and membrane:
current forced through ion channels
Look at single-channel currents, effects of internal and external
regulators
Not readily applicable to most endomembranes
Patch Clamp – The Primary Technique ForStudying Activity of Single Ion Channels
suck
pullSeal(cell-attached)
(whole cell)
(outside-out)
(inside-out)
pull
What Single Channel Currents Look Like
1 pA = 10-12 A; [Current: I]
Ions/s = I.N/F, where N = Avagadro’s No.; F = Faraday Const.
Here = 1.2 x 107 ions/s
Single channel recording only possible because of the relativelyhigh turnover rate
Conformational change between Open & Closed states known as
Gating
O
C
2pA
K+ channelfromT-lymphocyte
100 ms
Questions we can Answer from SingleChannel Recordings
1. What opens the channel?
Normally one (or more) of 3 factors:
• Voltage: changing membrane voltagein depolarizing (+ve) or hyperpolarizing (- ve)direction opening
• Neurotransmitter: binds and activatesfrom outside cell
• 2nd messenger: binds at cytosolicsurface and activates.
Typical recordings:
Note: No change in open channel current, just in time open:
An increase in open-state probability of the channel
(a) Non-permissiveconditions
(b) Permissiveconditions
openings rare
openings
frequent
O
C
(pA)t (ms)
O
C
2. Which ions flow through the channel? [Ion selectivity]Clamp voltage across membrane patch and measure open channelcurrent as a function of voltage:
Construct “current voltage” (I-V) relationship.
e.g.
Erev = –59 mV
Erev = 0 mV
-60 –40 –20 20 40 60
Conclude K+ ions are flowing through this channel
Measure reversalpotential (Erev)
Erev should correspond to equilibrium potential (Eion) of one of theions in solution
Eion = RT/zF ln([ion]out/[ion]in)
Example 1: [K+]out = 100 mM; [K+]in = 100 mM; EK = 0 mV = Erev
Example 2, [K+]out = 10 mM; [K+]in = 100 mM; EK = -59 mV = Erev
12
I (pA)
V (mV)
8
-8
3. What is the single channel conductance?
Ohm’s law: Voltage = Current . Resistance
V = I . R
Thus I = V. 1/R
Since 1/R = conductance (g),
the Slope of the I-V relationship = conductance
Unit of conductance: Siemens (S = 1/ohm)
e.g. previous slide: Slope = 8 pA/50 mV
= (8.10-12) / (50.10-3)
g = 160 pS
4. What inhibitors block the channel?Can either current when channel opens, or probability thatin open state.
e.g. tetrodotoxin (Na+ channels);
tetraethylammonium (K+ channels)
5. Does the channel inactivate?In continued presence of activating stimulus, openings lessfrequent
O
C
t
stimulus
Inactivation is a negative feedback mechanism to prevent toomuch channel activity
Channel type and channel function: anoverview
A. Voltage–Gated Channels
(i) K+ channels
Physiological role:
Primarily, stabilization of negative membrane potential(Vm)
Vm approaches EK if K+ conductance dominant
e.g. animal cell plasma membrane:
[K+]in = 150 mM, [K+]out = 5 mM
EK = 59 log ([K]out/[K+]in) = -87 mV
(ii) Ca2+ channels
Physiological role:
Ca2+ uptake into cell,
especially in Ca2+ -mediated stimulus-response coupling.
(iii) Na+ channels
Physiological role:
Always, to provide a depolarization:
The thrust of many action potentials
B. Neurotransmitter-gated channels
Neurotransmitter Ionic selectivity Effect of opening Response
Acetyl choline Cations Depolarization Action potential
Glutamate Cations Depolarization Action potential
-aminobutyric acid
(GABA) Cl- Hyperpol Inhibits a.p.
Glycine Cl- Hyperpol Inhibits a.p.
5-hydroxytryptamine
(5-HT) Cl- Hyperpol Inhibits a.p.
Cation channels are relatively non-selective among cations:physiologically carry mainly Na+ and Ca2+
C. Second messenger-gated channels
(i) Ca2+ - activated K+ channels
Restore membrane potential during Ca2+ signalling events
(ii) Cyclic nucleotide-gated (CNG) channels
Cation-selective: depolarizing signals in signal transduction:visual…… cGMP
olfactory… cAMP
(iii) Inositol 1,4,5-trisphosphate receptors (IP3R)
Ca2+-selective: release from ER during signal tranduction
Related to IP3Rs, on SR and ER ……
(iv) Ryanodine receptors RyR1 isoform interacts with p.m. Ca2+
channels in skeletal muscle
Other isoforms widespread: mediate Ca2+ release in cells: Ca2+-activated – give rise to Ca2+-induced Ca2+ release
Structure-Function Relations of IonChannels: Shaker-Type K+ Channels
Shaker class
K+: identified by chromosome walking in DrosophilaShaker mutant
Subsequently, related Ca2+ and Na+ channels purifiedfrom vertebrates by affinity chronatography with tight-binding inhibitors:
Channels are V-gated, and inactivate
Functional analysis: inject cRNA into Xenopusoocytes; measure currents after 3 – 4 days
Overall predicted domain structure of pore-forming (α) subunit from Hydropathy Analysis
K+ channel: 4 α subunits collectively form ion pore
Na+ and Ca2+ channels: each pore-forming subunit comprises 4α-likedomains
operate as monomers
S S S SS S1 2 3 4 5 6
N C
H5 or P loop
Ion Permeability and Selectivity
• The P loop forms the selectivity filter
A β –type 2o structure:
H5H5
H5
H5
S6
S6 S6
S5
S5
S5
S5
S6“Selectivity
filter”
Shaker K+ channel: sequence of P loop or H5 domain:
PD
AF
W
WA
VV
T M TT
VG
YG
DMTP
Evidence:• YG mutants lose ability to select for K+ over Na+
• A related Archeal channel has been crystallized: visualize K+
KcsA channel from Streptomyces lividans:Has only equivalent of S5, P loop, S6…
Drawing 9
“S5” “S6”
Aqueouspore
K+
ions
Ploop
Doyle et al. (1998) Science 405: 647
Pore with relativelyhydrophobic lining
+Helixwith dipole
2 K+ ions,mutually repulsive
K+ ionstabilizedby helicaldipoles
high turnover
Model for KcsA
K+ ions coordinatedby main-chaincarbonyl oxygens:high selectivity overNa+ (like valinomycin)
SF = selectivityfilter
Doyle et al. (1998) Science 405: 647
Voltage gating: voltage sensing is achieved by S4
S4 Sequence 'XXRXXRXXRXX(K/R)XXRXXKXX+ + + +
+
+
O
V
Sliding helix model
+ ve charges interact with – ve provided by other helices. Imposition of Vcases helix to swivel and project opening of gateSome evidence:Substitution of + ve residues changes voltage, but not permeation properties
+
Crystallization of an Archael Channel (KvAP)
has led to the Voltage Sensor Paddle Model
Proposes V-sensor is S4 + part of S3
All moves through membrane, pulling S5,S6 away from
interacting S5,S6 on other subunits
Evidence: Strategically-positioned Cys residues reacted withBiotin in side- & voltage-dependent fashion
Closed Open
Membrane
depol-arisation
Jiang et al. (2003) Nature 423: 33
Inactivation is achieved by a “Ball-and-Chain”
at N terminus
Can be either α or β subunit - depends on channel type
Mimics action of quaternary amine inhibitors
e.g. tetraethylammonium (TEA)
Drawing 12
Aldrich (2001) Nature 411: 643
First 10 residues: always hydrophobic – interact with poreResidues 11 – 21: always hydrophilic with net positive charge;interact with aqueous protein surfaces
Evidence: Some from deletion mutants…
Although 4 ball-and-chains, only one needed for blockage.
Singlechannelcurrents
inactivation
noinactivation
Zagotta et al. (1990) Nature 250: 568
Summary
1. The patch clamp technique is an informative method forstudying the properties of single ion channels.
2. Patch clamp allows study of single channel properties
a. Gating b. Ionic selectivity
c. Conductance d. Pharmacology
e. Inactivation
3. Channels fall into 3 main classes
a. V-gated b. Neurotransmitter-gated
c. Second messenger-gated
4. Shaker-type channels have been structurally characterised
Important function–structure correlates…
a. Ion permeation & selectivity: P loop
b. Voltage-sensing: S4 + part of S3
c. Inactivation: N-terminus ball-and-chain
Recommended