1. Cellular Neuroscience (207) Ian Parker Lecture #3 - Voltage- and ligand- gated ion channels

2. Diversity of ion channels Ion channels can be categorized by two main properties - 1.Their gating properties; i.e. what makes them open. Major categories are voltage- and ligand-gated channels (neurotransmitters and second messengers). Other channels are gated by temperature, mechanical stress. 2.Their permeability properties; i.e. what ions pass through the open channel, and what electrical current results from this.

3. Ion permeability Na + (inward current) K + (outward current) Ca 2+ (inward current) Cl - (usually outward current) Upstroke of action potential Rapid repolarization during action potential Control of excitability / interspike interval Electrical signal: i.e. depolarization (Ca 2+ action potential) Chemical signal: rise in intracellular [Ca 2+ ], e.g. opening of Ca 2+ -dependent K + channels Synaptic inhibition

4. Voltage-gated channels – nomenclature and diversity Named by the ion that goes through them :e.g. sodium channel. (different to nomenclature for ligand-gated channels, which are usually named for their ligand : e.g. GABA receptor) Old nomenclature was arbitrary, based on functional distinctions (e.g. A-type and K-type K + channels), or on gene mutations from which channels first cloned (e.g. shaker and shaw K + channels) New, systematic (but boring) nomenclature and classification based on ‘family tree’ of sequence homology in channel genes (e.g. Ca V 1.3, K V 3.1) For illustration – you don’t have to memorize this!

5. All Na + channels have similar properties, whereas K + channels are highly diverse Similar inactivation kinetics of Na + currents from many species/organs - Na channels have only one, stereotyped job; to make the action potential go up. Different K + channels show very different inactivation kinetics – they serve many different purposes

6. Relation between single channel currents and whole-cell current A. Inactivating Na channels give transient whole-cell current Questions….. What determines peak whole-cell current? What determines rise-time of whole-cell current? What determines the decay rate of whole- cell current?

7. Relation between single channel currents and whole-cell current B. A. Non-inactivating K channels give sustained whole-cell current Questions….. What determines mean steady-state whole-cell current? What determines rise-time of whole cell current? Why is current whole-cell current trace ‘noisier’ during depolarization? Might this tell us something?

8. Mechanism of voltage-dependent activation – gating charge movement S4 region of channel contains highly charged amino acids, and physically moves in response to voltage change. This causes opening of channel (but we don’t yet know how). Movement of S4 exposes residues to extracellular solution, and generates a ‘gating current’, which can be measured. Mutations in S4 that reduce the # of charges reduce the gating current and make the voltage dependence of Na conductance (i.e. probability of channel opening) a less steep function of voltage.

9. Channel inactivation – ‘ball and chain’ model for inactivation of Shaker K + channel Inactivation – channel stops passing current, even with maintained depolarization. Mechanism involves a ‘gate’ different to that controlling activation. ‘Ball and chain’ is one (but not the only) mechanism. Peptide ‘ball’ on flexible tether (all parts of K channel subunit) swings in to block channel soon after it opens. Test of model. Mutation of shaker channel that deletes ball removes activation. Can then recover inactivation by separately expressing peptide balls, even though these are no longer tethered to the channel

10. Ligand-gated channels : the nicotinic ACh receptor as an exemplar Receptor/channel molecule comprised from total of 5 subunits – 2x , 1each  Channel opening requires  that 2 ACh molecules be bound simultaneously to the 2  subunits. Channel closes when one ACh dissociates. Mean channel lifetime is thus a function of mean time for which agonist stays bound. This is a function both of the receptor and the agonist – e.g. carbachol gives shorter mean open time than ACh.

11. Requirement for binding of 2 ACh molecules means that channel opening increases as square of [ACh] Low agonist concentration Double agonist concentration Channel openings become much more frequent with increasing [agonist]. Mean open time does not change with [agonist] Mean channel closed time becomes much shorter : i.e. frequency of openings increases

12. Hill coefficient reveals degree of cooperativity : i.e. number of agonist molecules required to cause channel opening Log reciprocal mean closed / open time Closed time shortens with slope of 2 on log/log plot (i.e. as square of agonist concentration) Mean open lifetime does not change with [agonist] – it depends on agonist unbinding, not binding. [agonist] at which lines cross (i.e. when mean open time = closed time) gives measure of apparent affinity of agonist A double log plot causes power functions (square, cube etc.) to appear as straight lines. The slope of the line (Hill coefficient) indicates the power: e.g. square = slope of 2, cube = 3, etc. Agonist concentration

13. Other kinetic features 1.‘Nachschlags’ – brief closings during chanel openings 2. Desensitization bursts – whole-cell current declines even in sustained presence of agonist Agonist application Whole cell current declines Individual channels show ‘bursts’ of openings, interrupted by long silent intervals when channel is desensitized. Whole cell current declines as more channels enter desensitized state.

14. A (simplified) kinetic model of channel gating A + RAR +AA2RA2RA 2 R* A 2 D Agonist (ACh) Receptor (channel shut) Receptor (channel open) Desensitized receptor (channel shut) Receptor can exist in 5 states: each with a characteristic mean lifetime Only 1 open state (A2R*) – so distribution of open times shows single exponential. But 4 closed states – so closed time distribution is actually made up of 4 exponential components. Of these A2R (flickers) and A2D (silent intervals during desensitization) are independent of [agonist]: Lifetimes of R and AR shorten with increasing [agonist]