Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker.
Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a "gate" in the transmembrane domain (TMD). We used Phi-value analysis to probe the relative timing of the gating motions of alpha-subunit residues located near the ECD-TMD interface. Mutation of four of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (K(eq)) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Phi-value for the whole linker was approximately 0.64. One interpretation of this result is that the gating motions of the M2-M3 linker are approximately synchronous with those of much of M2 (approximately 0.64), but occur after those of the transmitter binding site region (approximately 0.93) and loops 2 and 7 (approximately 0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed K(eq) by 2800-, 10-, and 18-fold, respectively, and with an average Phi-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (< or =0.51 kcal mol(-1)). The M2-M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an approximately 16-A border and involve about a dozen residues.
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, USA.
Archana
A
Jha
David J
DJ
Cadugan
Prasad
P
Purohit
Anthony
A
Auerbach
D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D004735:Energy Transfer; D005110:Extracellular Space / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014101:Torpedo
D018076:DNA, Complementary; D011950:Receptors, Cholinergic