Tryptophan Substitutions Reveal the Role of Nicotinic Acetylcholine Receptor α-TM3 Domain in Channel Gating: Differences between Torpedo and Muscle-Type AChR

Manuel Navedo, Madeline Nieves, Legier Rojas, Jose A. Lasalde-Dominicci

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

A recent tryptophan scanning of the α-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A., Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here we extend the study of the α-TM3 domain to the muscle-type AChR by examining functional consequences of single tryptophan substitutions at five conserved positions (αM282, αF284, αV285, αA287, and αI290) homologous to the α-TM3 positions that were recently characterized in the Torpedo AChR. Similarly to the Torpedo AChR, mutations αM282W and αV285W, which are presumed to face the interior of the protein, did not exhibit functional channel activity. Nevertheless, significant expression levels of these mutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR, tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalized macroscopic response. Single-channel recordings at low ACh concentration revealed that the increase in AChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration. These results suggest that tryptophan substitution directly affects channel gating, primarily the channel closing rate. Our results suggest that residues facing the interior of the protein (i.e., αM282 and αV285) may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations (i.e., F284W and I290W) presumably facing the lipid environment display a very different functional response between these two AChR species.

Original languageEnglish (US)
Pages (from-to)78-84
Number of pages7
JournalBiochemistry
Volume43
Issue number1
StatePublished - Jan 13 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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