Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics.
The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of alphaM4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in alphaM4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing alphaC418A and alphaT422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type alpha/alphaT422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the closing process by approximately 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in alphaM4 in AChR channel gating mechanism.
Bouzat
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C
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, 8000 Bahía Blanca, Argentina. inbouzat@criba.edu.ar
Roccamo
A M
AM
Garbus
I
I
Barrantes
F J
FJ
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D015640:Ion Channel Gating / Q000235:genetics*; D008051:Lipid Bilayers / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D043588:Receptor, Muscarinic M4; D011950:Receptors, Cholinergic / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D008051:Lipid Bilayers; D043588:Receptor, Muscarinic M4; D011950:Receptors, Cholinergic; D011976:Receptors, Muscarinic; D000409:Alanine