Kinetic, mechanistic, and structural aspects of unliganded gating of acetylcholine receptor channels - A single-channel study of second transmembranesegment 12 ' mutants
C. Grosman et A. Auerbach, Kinetic, mechanistic, and structural aspects of unliganded gating of acetylcholine receptor channels - A single-channel study of second transmembranesegment 12 ' mutants, J GEN PHYSL, 115(5), 2000, pp. 621-635
The spontaneous activity of adult mouse muscle acetylcholine receptor chann
els, transiently expressed in HEK-293 cells, was studied with the patch-cla
mp technique. To increase the frequency of unliganded openings, mutations a
t the 12' position of the second transmembrane segment were engineered. Our
results indicate that: (a) in both wild type and mutants, a C <-> O kineti
c scheme provides a good description of spontaneous gating. In the case of
some mutant constructs, however additional states were needed to improve th
e fit to the data. Similar additional states were also needed in one of su;
patches containing wild-type acetylcholine receptor channels; (b) the delt
a 12' residue makes a more pronounced contribution to unliganded gating tha
n the homologous residues of the alpha, beta, and epsilon subunits; (c) com
binations of second transmembrane segment 12' mutations in the four differe
nt subunits appear to have cumulative effects; (d) the volume of the side c
hain at delta 12' is relevant because residues larger than the wild-type Se
r increase spontaneous gating; (e) the voltage dependence of the unliganded
gating equilibrium constant is the same as that of diliganded gating, but
the voltage dependences of the opening and closing rate constants are oppos
ite (this indicates that the reaction pathway connecting the closed and ope
n states of the receptor changes upon ligation); (f) engineering binding-si
te mutations that decrease diliganded gating (alpha Y93F, alpha YI90W, and
alpha D200N) reduces spontaneous activity as well (this suggests that even
in the absence of ligand the opening of the channel is accompanied by a con
formational change at the binding sites); and (g) the diliganded gating equ
ilibrium constant is also increased by the 12' mutations. Such increase is
independent of the particular ligand used as the agonist, which suggests th
at these mutations affect mostly the isomerization step, having little, if
any, effect on the ligand-affinity ratio.