1. Cellular Neuroscience (207) Ian Parker Lecture # 6 - Ligand-gated channels http://parkerlab.bio.uci.edu

2. The synapse that has taught us most of what we know about synaptic transmission The frog neuromuscular junction (‘endplate’) Advantages: the postsynaptic cell (muscle) is big (100  m x 2 cm) so easy to impale with microelectrode; although the presynaptic axon is very thin, the nerve is easily dissected for extracellular stimulation Could electrical transmission work at an endplate? Membrane area of nerve terminal = 2 x 10-5 cm2 Assuming Na current density of 1 mA cm-2, then total current = 2 x 10-8 A Input resistance of muscle fiber ~ 10^5 Ohm So, nerve terminal could depolarize the muscle by only ~ 2mV NOT ENOUGH

3. Presynaptic release of neurotransmitter (acetylcholine : ACh) causes opening of ligand-gated channels (AChR), leading to the generation of the endplate potential

4. Kinetic scheme for agonist-gating : exemplified by nicotinic ACh receptor 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 longer mean open time than ACh.

5. 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

6. 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

7. 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.

8. 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]