Transcript Ion permeability - The Parker Lab at UCI

Cellular Neuroscience (207)
Ian Parker
Lecture #3 - Voltage- and ligandgated ion channels
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.
• Ion permeability
Na+ (inward current)
Upstroke of action potential
Rapid repolarization during action potential
K+ (outward current)
Control of excitability / interspike interval
Ca2+ (inward current)
Electrical signal: i.e. depolarization
(Ca2+ action potential)
Chemical signal: rise in intracellular
[Ca2+], e.g. opening of Ca2+-dependent
K+ channels
Cl- (usually outward
current)
Synaptic inhibition
Voltage-gated channels – nomenclature and
diversity
•
Named by the ion that goes through them :e.g. sodium channel.
•
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)
(different to
nomenclature for ligand-gated channels, which are usually named for their ligand : e.g.
GABA receptor)
New, systematic (but boring)
nomenclature and classification
based on ‘family tree’ of sequence
homology in channel genes (e.g.
CaV1.3, KV3.1)
For illustration – you don’t have to memorize this!
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
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 wholecell current?
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?
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.
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
Ligand-gated channels : the nicotinic ACh
receptor as an exemplar
Receptor/channel molecule
comprised from total of 5
subunits – 2x a, 1each b,g,d.
Channel opening requires that 2
ACh molecules be bound
simultaneously to the 2 a
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.
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
Hill coefficient reveals degree of cooperativity : i.e. number
of agonist molecules required to cause channel opening
Log reciprocal mean closed / open time
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
Closed time shortens with slope of 2 on
log/log plot (i.e. as square of agonist
concentration)
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 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.
A (simplified) kinetic model of channel gating
Agonist (ACh)
A+R
Receptor
(channel shut)
AR +A
A2R
A2 R*
Receptor
(channel open)
A2 D
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]