Cis-trans 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 superfamily. 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), 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.
Lummis
Sarah C R
SC
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
Beene
Darren L
DL
Lee
Lori W
LW
Lester
Henry A
HA
Broadhurst
R William
RW
Dougherty
Dennis A
DA
D000818:Animals; D045744:Cell Line, Tumor; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000187:drug effects*; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D009447:Neuroblastoma; D018377:Neurotransmitter Agents / Q000378:metabolism / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism; D011392:Proline / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary / Q000187:drug effects; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007473:Ion Channels; D018377:Neurotransmitter Agents; D044406:Receptors, Serotonin, 5-HT3; D011392:Proline