Cellular Neuroscience (207)Ian Parker

Lecture # 4 - Ligand-gated channels

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 µm 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 cm2Assuming Na current density of 1 mA cm-2, then total current = 2 x 10-8 AInput resistance of muscle fiber ~ 10^5 OhmSo, nerve terminal could depolarize the muscle by only ~ 2mV NOT ENOUGH

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

potential

Kinetic scheme for agonist-gating : exemplified by nicotinic ACh receptor

Receptor/channel molecule comprised from total of 5 subunits – 2x α, 1each β,γ,δ.

Channel opening requires that 2 ACh molecules be bound simultaneously to the 2 α 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.

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 timebecomes much shorter : i.e. frequency of openings increases

Hill coefficient reveals degree of cooperativity : i.e. number of agonist molecules required to cause channel opening

Log

reci

proc

al m

ean

clos

ed /

ope

n tim

e

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

Other kinetic features

1. ‘Nachschlags’ – brief closingsduring 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.

A (simplified) kinetic model of channel gating

A + R AR +A A2R A2 R* A2 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]