Chemistry Reference
In-Depth Information
7.5
Modulation of Ion Channel Current by Ligand Binding
7.5.1
Ligand-activated Ion Channels: Decoupling Ligand Binding and Channel Gating with
Single-molecule Patch-clamp
Ligand-gated ion channels switch between a closed (non-conducting) conformation
and an open (conducting) conformation. In most cases, ligand binding increases the
open probability due to higher af
nity to the open state, which mainly arises from a
smaller dissociation rate constant from the open channel. A simpli
ed allosteric
model that separates the ligand-binding step from the channel gating step is usually
represented by the mechanism described by del Castillo and Katz [72]:
k
ass
!
k
diss
LR
b
LR
|
{z
}
gating
!
a
R
ð
7
:
6
Þ
|
{z
}
binding
where R represents a closed channel, R
an open channel, L denotes a ligand (agonist
molecule), k
ass
and k
diss
are the association and dissociation rate constants,
b
the closing and opening rate constants of the channel, respectively. This model
assumes fast equilibrium binding versus the subsequent conformational changes
occurring during channel gating.
Single-channel patch-clamp recordings revealed the
fine structure of the effective
openings, referred to as bursts, composed of transient closing events. This allowed
the evaluation of the binding and gating rate constants separately [73]. The rate
constants k
diss
,
a
and
, referring to the liganded receptor, can be accurately estimated
by using different methods. Because the association reaction is not usually diffusion
limited and the number of channels present in the patch of membrane cannot be
unequivocally de
ned [73], single-channel current measurement is not however the
most reliable approach to determine k
ass
.
a
and
b
7.5.2
The Nicotinic Acetylcholine Receptor as a Prototypical Example
The nicotinic acetylcholine receptor (nAChR) is a heteropentameric, cation-selective
channel that is activated by binding of the natural neurotransmitter acetylcholine.
Two ligand binding sites are located at subunit interfaces in the large extracellular
domain. Themuscle type nAChR is concentrated at neuromuscular junctions, where
it mediates fast synaptic transmission by depolarizing the post-synaptic membrane.
Single-channel current recordings frompatches of end-platemembranes activated by
acetylcholine revealed a two-state reaction scheme, which re
ects transient openings
of nAChR channels. Burst analysis of elementary currents allowed the derivation of
an activation mechanism with microscopic binding and gating rate constants.
Figure 7.2A shows single-channel currents simulated with a two-binding site
mechanism in which only the diliganded receptor initiates channel opening as
