Module 0220502 Membrane Biogenesis and Transport Lecture 15 Ion Channels Dale Sanders 9 March 2009
Aims: By the end of the lecture you should understand The principles behind the patch clamp technique; What information about ion channels can be extracted from single channel recordings; What are the main classes of ion channel in biology, and what they do; How structure relates to function in the Shaker class of ion channel: permeability, voltage-sensing and inactivation.
Reading Lodish et al (2008) Molecular Cell Biology, 6 th ed. pp.458-465 & 1006-1013 More detailed, highly readable original papers, all from the lab of Rod MacKinnon: 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 the inactivation gate and quaternary amine inhibitors. Nature 411: 657
Patch Clamp The Primary Technique For Studying Activity of Single Ion Channels Seal (cell-attached) pull (inside-out) suck (whole cell) pull (outside-out) 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
What Single Channel Currents Look Like 2pA O C K + channel from T-lymphocyte 100 ms 1 pa = 10-12 A; [Current: I] Ions/s = I.N/F, where N = Avagadro s No.; F = Faraday Const. Here = 1.2 x 10 7 ions/s Single channel recording only possible because of the relatively high turnover rate Conformational change between Open & Closed states known as Gating
Questions we can Answer from Single Channel Recordings 1. What opens the channel? Normally one (or more) of 3 factors: Voltage: changing membrane voltage in depolarizing (+ve) or hyperpolarizing (- ve) direction opening Neurotransmitter: binds and activates from outside cell 2 nd messenger: binds at cytosolic surface and activates.
Typical recordings: (a) Non-permissive conditions O C openings rare (b) Permissive conditions (pa) t (ms) O C openings frequent Note: No change in open channel current, just in time open: An increase in open-state probability of the channel
2. Which ions flow through the channel? [Ion selectivity] Clamp voltage across membrane patch and measure open channel current as a function of voltage: Construct current voltage (I-V) relationship. e.g. E rev = 59 mv -60 40 20 20 40 60 V (mv) E rev = 0 mv Measure reversal potential (E rev ) E rev should correspond to equilibrium potential (E ion ) of one of the ions in solution E ion = RT/zF ln([ion] out /[ion] in ) I (pa) 2 Example 1: [K + ] out = 100 mm; [K + ] in = 100 mm; E K = 0 mv = E rev Example 2, [K + ] out = 10 mm; [K + ] in = 100 mm; E K = -59 mv = E rev Conclude K + ions are flowing through this channel 8-8 1
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 that in open state. e.g. tetrodotoxin (Na + channels); tetraethylammonium (K + channels) 5. Does the channel inactivate? In continued presence of activating stimulus, openings less frequent stimulus t Inactivation is a negative feedback mechanism to prevent too much channel activity O C
Channel type and channel function: an overview A. Voltage Gated Channels (i) K + channels Physiological role: Primarily, stabilization of negative membrane potential (V m ) V m approaches E K if K + conductance dominant e.g. animal cell plasma membrane: [K + ] in = 150 mm, [K + ] out = 5 mm E K = 59 log ([K] out /[K + ] in ) = -87 mv
(ii) Ca 2+ channels Physiological role: Ca 2+ uptake into cell, especially in Ca 2+ -mediated stimulusresponse 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 Ca 2+
C. Second messenger-gated channels (i) Ca 2+ - activated K + channels Restore membrane potential during Ca 2+ 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 (IP 3 R) Ca 2+ -selective: release from ER during signal tranduction Related to IP 3 Rs, on SR and ER (iv) Ryanodine receptors RyR1 isoform interacts with p.m. Ca 2+ channels in skeletal muscle Other isoforms widespread: mediate Ca 2+ release in cells: Ca 2+ - activated give rise to Ca 2+ -induced Ca 2+ release
Structure-Function Relations of Ion Channels: Shaker-Type K + Channels Shaker class K + : identified by chromosome walking in Drosophila Shaker mutant Subsequently, related Ca 2+ and Na + channels purified from vertebrates by affinity chronatography with tightbinding inhibitors: Channels are V-gated, and inactivate Functional analysis: inject crna into Xenopus oocytes; measure currents after 3 4 days
Overall predicted domain structure of poreforming (α) subunit from Hydropathy Analysis H5 or P loop S S S SS S 1 2 3 4 5 6 N C K + channel: 4 α subunits collectively form ion pore Na + and Ca 2+ channels: each pore-forming subunit comprises 4α-like domains operate as monomers
Ion Permeability and Selectivity The P loop forms the selectivity filter A β type 2 o structure: Shaker K+ channel: sequence of P loop or H5 domain: Selectivity filter S5 S6 S6 Evidence: H5 H5 S5 S5 H5 H5 S6 S6 S5 P P D T A M F D W W Y G G AV V T M T T V 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 S5 S6 P Drawing 9 loop K+ ions Aqueous pore Doyle et al. (1998) Science 405: 647
Model for KcsA SF = selectivity filter + 2 K+ ions, mutually repulsive Helix with dipole K+ ion stabilized by helical dipoles high turnover Pore with relatively hydrophobic lining K+ ions coordinated by main-chain carbonyl oxygens: high selectivity over Na+ (like valinomycin) Doyle et al. (1998) Science 405: 647
Voltage gating: voltage sensing is achieved by S4 S4 Sequence 'XXRXXRXXRXX(K/R)XXRXXKXX + + + + + + Sliding helix model O + V + ve charges interact with ve provided by other helices. Imposition of V cases helix to swivel and project opening of gate Some 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 Membrane depolarisation Open Closed 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 with Biotin in side- & voltage-dependent fashion 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 pore Residues 11 21: always hydrophilic with net positive charge; interact with aqueous protein surfaces Evidence: Some from deletion mutants inactivation Single channel currents no inactivation Although 4 ball-and-chains, only one needed for blockage. Zagotta et al. (1990) Nature 250: 568
Summary 1. The patch clamp technique is an informative method for studying 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. c. Second messenger-gated Neurotransmitter-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