Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservat...
| Autores: | , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2004 |
| País: | Argentina |
| Institución: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repositorio: | CONICET Digital (CONICET) |
| Idioma: | inglés |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/53089 |
| Acceso en línea: | http://hdl.handle.net/11336/53089 |
| Access Level: | acceso abierto |
| Palabra clave: | Acetylcholine Acetylcholine Receptor Ion Channel Patch Clamp Site-Directed Mutagenesis https://purl.org/becyt/ford/3.3 https://purl.org/becyt/ford/3 |
| Sumario: | The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus. |
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