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Cistrans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel


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 cistrans 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 cistrans energy gap of the proline analogue and the activation of the channel, suggesting that cistrans 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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Figure 1: Overall layout of the 5-HT 3 receptor showing the extracellular (predominantly β-sheet) and transmembrane (α-helical) regions.
Figure 2: Unnatural amino acid mutagenesis.
Figure 3: Current traces in voltage-clamp experiments (at approximately EC 50 and approximately I max ) for receptors expressing unnatural amino acids.
Figure 4: 1 H NMR of a model peptide.
Figure 5: A proposed gating mechanism.


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S.C.R.L. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Science. The work at Caltech was supported by the National Institutes of Health. We thank K. L. Price and A. J. Thompson for assistance with modelling.

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Correspondence to Dennis A. Dougherty.

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Lummis, S., Beene, D., Lee, L. et al. Cistrans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel. Nature 438, 248–252 (2005).

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