Kinetic, mechanistic, and structural aspects of unliganded gating of acetylcholine receptor channels - A single-channel study of second transmembranesegment 12 ' mutants

Citation
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
Citations number
62
Categorie Soggetti
Physiology
Journal title
JOURNAL OF GENERAL PHYSIOLOGY
ISSN journal
00221295 → ACNP
Volume
115
Issue
5
Year of publication
2000
Pages
621 - 635
Database
ISI
SICI code
0022-1295(200005)115:5<621:KMASAO>2.0.ZU;2-O
Abstract
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.