Lummis, S. C. R. et al. Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel. Nature 438, 248-252

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.
Nature (Impact Factor: 41.46). 12/2005; 438(7065):248-52. DOI: 10.1038/nature04130
Source: PubMed


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.

Download full-text


Available from: Richard W Broadhurst,
  • Source
    • "Agonist binding induces rigid body motions, which are translated into transient movements of the pore lining M2 a helices of the transmembrane domain (TMD) by a series of loops at the ECD/TMD interface (Althoff et al., 2014; Sauguet et al., 2014; Unwin and Fujiyoshi, 2012). Considerable attention has focused on elucidating the gating movements of these interfacial loops, which form the primary allosteric path leading from the agonist site to the channel gate (Grutter et al., 2005; Jha et al., 2007; Lee and Sine, 2005; Lummis et al., 2005). In contrast, structures not directly involved in the primary allosteric path have received less attention, even though a number of allosteric modulators influence gating via these auxiliary sites (Figure 1A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The gating of pentameric ligand-gated ion channels is sensitive to a variety of allosteric modulators that act on structures peripheral to those involved in the allosteric pathway leading from the agonist site to the channel gate. One such structure, the lipid-exposed transmembrane α helix, M4, is the target of lipids, neurosteroids, and disease-causing mutations. Here we show that M4 interactions with the adjacent transmembrane α helices, M1 and M3, modulate pLGIC function. Enhanced M4 interactions promote channel function while ineffective interactions reduce channel function. The interface chemistry governs the intrinsic strength of M4-M1/M3 inter-helical interactions, both influencing channel gating and imparting distinct susceptibilities to the potentiating effects of a lipid-facing M4 congenital myasthenic syndrome mutation. Through aromatic substitutions, functional studies, and molecular dynamics simulations, we elucidate a mechanism by which M4 modulates channel function. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 07/2015; 23(9). DOI:10.1016/j.str.2015.06.020 · 5.62 Impact Factor
  • Source
    • "Reorganization of transmembrane helices and rigid body rotation of an extracellular domain are the important changes observed in the open and closed state structures of pentameric ligand-gated ion channels [26]. Using a series of proline analogs, experiments have suggested that cis–trans isomerization of a single proline residue provides a molecular switch for inter-converting open and closed states in the channels formed by 5HT3 receptors which are members of Cys-loop receptor superfamily [27]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10-15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 03/2015; 1848(6). DOI:10.1016/j.bbamem.2015.03.013 · 4.66 Impact Factor
  • Source
    • "In the process of amyloid fibril formation they are important, both, in the nucleation and in fibril elongation [26] [27]. The cis/trans isomerization of the peptide bond preceding proline is considered to form an intrinsic molecular switch controlling several physiologically relevant processes, such as opening of the pore of a neurotransmitter-gated ion channel [28] [29]. Peptidyl prolyl cis/trans isomerases (PPIases) are enzymes that catalyze the cis/ trans isomerization of prolyl bonds. "

Show more