1. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK
2. Division of Chemistry and Chemical Engineering, and
3. Division of Biology, California Institute of Technology, Pasadena, California 91125, USA
4. *These authors contributed equally to this work

Correspondence to: Dennis A. Dougherty² Correspondence and requests for materials should be addressed to D.A.D. (Email: dadougherty@caltech.edu).

Abstract

5-Hydroxytryptamine type 3 (5-HT₃) receptors are members of the Cys-loop receptor superfamily¹. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2–M3)^{2, 3}, can link binding to gating through a cis–trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis–trans energy gap of the proline analogue and the activation of the channel, suggesting that cis–trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2–M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2–M3 loop and the critical role of Pro 8* in the 5-HT₃ receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.

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