Nicotinic receptor M3 transmembrane domain: position 8' contributes to channel gating.
The nicotinic acetylcholine receptor (nAChR) is a pentamer of homologous subunits with composition alpha(2)(beta)(epsilon)(delta) in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 8' of the M3 domain is phenylalanine in all heteromeric alpha subunits, whereas it is a hydrophobic nonaromatic residue in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle nAChR activation by combining mutagenesis with single-channel kinetic analysis. Construction of nAChRs carrying different numbers of phenylalanine residues at 8' reveals that the mean open time decreases as a function of the number of phenylalanine residues. Thus, all subunits contribute through this position independently and additively to the channel closing rate. The impairment of channel opening increases when the number of phenylalanine residues at 8' increases from two (wild-type nAChR) to five. The gating equilibrium constant of the latter mutant nAChR is 13-fold lower than that of the wild-type nAChR. The replacement of (alpha)F8', (beta)L8', (delta)L8', and (epsilon)V8' by a series of hydrophobic amino acids reveals that the structural bases of the observed kinetic effects are nonequivalent among subunits. In the alpha subunit, hydrophobic amino acids at 8' lead to prolonged channel lifetimes, whereas they lead either to normal kinetics (delta and epsilon subunits) or impaired channel gating (beta subunit) in the non-alpha subunits. The overall results indicate that 8' positions of the M3 domains of all subunits contribute to channel gating.
De Rosa
María José
MJ
Instituto de Investigaciones Bioquímicas, Universidad Nacíonal del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, Bahía Blanca, Argentina.
Rayes
Diego
D
Spitzmaul
Guillermo
G
Bouzat
Cecilia
C
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004734:Energy Metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D010649:Phenylalanine