structure, but with someb-structure. Eacha-helical region has been designated
TM1–TM4 and the experimental evidence supports the view that the TM2 regions of
each of the five subunits line the ion channel with the TM1 and TM3 regions forming a
scaffold to support the channel. Binding studies have provided evidence for the
allosteric binding of two molecules of agonist with a Hill constant of about 2. Affinity
labelling studies have shown that the ligand-binding sites are in a cleft between two
subunits. In both cases ana-subunit has the principal role in binding with thegandd
subunits playing a minor role. The first ligand molecule binds to thea 1 subunit which
is in contact with thedsubunit and the second ligand molecule toa 2 which is in
contact with thegsubunit. Thus the two binding sites are not structurally identical.
Genetically engineered variants of the five subunits have shown that the absence of
theasubunit results in the lack of binding of acetylcholine thereby confirming the
importance of this subunit.
Electron microscopic study of the nAChR on theTorpedoelectric organ postsynap-
tic membrane has given an indication of the three-dimensional structure of the
channel. It is funnel-shaped with a large proportion of the receptor outside the
membrane protruding into the postsynaptic cleft. The channel is 25–30 A ̊ wide at
the entrance and only 6.4 A ̊wide at its narrowest point. Three rings of negatively
charged amino acid residues, all on the four TM2 helices, line the narrow part of the
channel and appear to determine its selectivity. The importance of these amino acid
residues has been confirmed by mutagenicity studies. Detailed electrophysiological
studies have revealed that channel opening involves the subtle rearrangement of three
transmembranea-helices, one in each of the TM1, TM2 and TM3 segments. The whole
process is kinetically complex but the essential features may be represented as
follows:AþR Ð ARþA Ð A 2 R Ð A 2 R Ð A 2 R
channel: closed closed closed open desensitisedwhere A is acetylcholine and R the receptor containing two binding sites for acetyl-
choline, one on eachasubunit.
Measurement of the numerous rate constants for these reversible processes has
revealed that the rate constant for the opening of the channel is greater than the rate
constants for the corresponding reverse process (i.e. the reversion to the closed
conformation), for the dissociation of a ligand molecule from the closed conformation
and for the transition from the open active state to the desensitised state. The conse-
quence of this is that many opening and closing events of the channel occur before
either the transition to the desensitised state or a molecule of ligand dissociates from
the binding site. The mean channel open time for torpedo nAChR is 3.0 ms and
the mean closed time 94ms within the bursts of opening and closing activity. The
desensitised receptor (R**) eventually reverts to the closed resting state (R).
Desensitisation may be linked to phosphorylation. All five subunits contain amino
acid residues located between the TM3 and TM4 regions that are potential sites for
phosphorylation, and phosphorylation of the receptor has been shown to occur at two
serine residues on each of theganddsubunits. Each phosphate group introduces two
negative oxygen atoms that could induce important conformational changes in the689 17.4 Mechanisms of signal transduction