Tryptophan scanning of the acetylcholine receptor's betaM4 transmembrane domain: decoding allosteric linkage at the lipid-protein interface with ion-channel gating.
The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that mediates fast excitatory synaptic transmission in the peripheral and central nervous systems. Changes in the structure and function of the AChR can lead to serious impairment of physiological processes. In this study, we combined site-directed mutagenesis, radioligand binding assays, electrophysiological recordings and Fourier analyses to characterize the functional role and structural aspects of the betaM4 transmembrane domain of the Torpedo AChR. We performed tryptophan replacements, from residues L438 through F455, along the betaM4 transmembrane domain. Expression levels of mutants F439W-G450W and F452W-I454W produced peak currents similar to or lower than those in wild-type (WT). Tryptophan substitutions at positions L438 and T451 led to a deficiency in either subunit expression or receptor assembly. Mutations L440W, V442W, C447W and S453W produced a gain-of-function response. Mutation F455W produced a loss of ion channel function. The periodicity profile of the normalized expression level (closed state) and EC(50) (open state) revealed a minor conformational change of 0.4 residues/turn of the betaM4 domain. These findings suggest that a minor movement of the betaM4 domain occurs during channel activation.
Díaz-De León
Rosedelma
R
Department of Biology, University of Puerto Rico, San Juan, Puerto Rico 00931-3360.
Otero-Cruz
José David
JD
Torres-Nuñez
David Abner
DA
Casiano
Anette
A
Lasalde-Dominicci
José Antonio
JA
D000495:Allosteric Site; D000818:Animals; D004594:Electrophysiology; D006801:Humans; D008055:Lipids; D009068:Movement; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011506:Proteins; D011869:Radioligand Assay; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014364:Tryptophan
D008055:Lipids; D011506:Proteins; D011950:Receptors, Cholinergic; D014364:Tryptophan