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.

[1]  P. Schofield,et al.  The Surface Accessibility of the Glycine Receptor M2–M3 Loop Is Increased in the Channel Open State , 2001, The Journal of Neuroscience.

[2]  E. Kirkness,et al.  A cytoplasmic region determines single-channel conductance in 5-HT3 receptors , 2003, Nature.

[3]  J. Priestley,et al.  Antibodies against the extracellular domain of the 5-HT3 receptor label both native and recombinant receptors. , 1999, Brain research. Molecular brain research.

[4]  D. Colquhoun,et al.  Binding, gating, affinity and efficacy: The interpretation of structure‐activity relationships for agonists and of the effects of mutating receptors , 1998, British journal of pharmacology.

[5]  G P Hess,et al.  Inhibition of the serotonin 5-HT3 receptor by nicotine, cocaine, and fluoxetine investigated by rapid chemical kinetic techniques. , 2001, Biochemistry.

[6]  D. Kern,et al.  Rotational Barriers of cis/trans Isomerization of Proline Analogues and Their Catalysis by Cyclophilin§ , 1997 .

[7]  A. Auerbach,et al.  The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element , 2000, The Journal of general physiology.

[8]  Henry A Lester,et al.  Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamine3 Receptor Revealed by Unnatural Amino Acid Mutagenesis , 2004, The Journal of Neuroscience.

[9]  H. Lester,et al.  Probing the role of a conserved M1 proline residue in 5-hydroxytryptamine(3) receptor gating. , 2000, Molecular pharmacology.

[10]  Y. Fujiyoshi,et al.  Structure and gating mechanism of the acetylcholine receptor pore , 2003, Nature.

[11]  William D. Lubell,et al.  Use of Steric Interactions To Control Peptide Turn Geometry. Synthesis of Type VI beta-Turn Mimics with 5-tert-Butylproline. , 1999, The Journal of organic chemistry.

[12]  H. Scheraga,et al.  Retention of the cis proline conformation in tripeptide fragments of bovine pancreatic ribonuclease A containing a non-natural proline analogue, 5,5-dimethylproline , 1999 .

[13]  T. Sixma,et al.  Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors , 2001, Nature.

[14]  Henry A. Lester,et al.  From ab initio quantum mechanics to molecular neurobiology: A cation-p binding site in the nicotinic receptor (nicotinic acetylcholine receptorycation-p interactionyunnatural amino acids) , 1998 .

[15]  Mark S.P. Sansom,et al.  Hinges, swivels and switches: the role of prolines in signalling via transmembrane α-helices , 2000 .

[16]  H. Lester,et al.  Backbone Mutations in Transmembrane Domains of a Ligand-Gated Ion Channel Implications for the Mechanism of Gating , 1999, Cell.

[17]  C. Deber,et al.  Hypothesis about the function of membrane-buried proline residues in transport proteins. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. Lummis,et al.  The transmembrane domain of the 5-HT3 receptor: its role in selectivity and gating. , 2004, Biochemical Society transactions.

[19]  R. Broadhurst,et al.  Structure and properties of a dimeric N-terminal fragment of human ubiquitin. , 2001, Journal of molecular biology.

[20]  N. Unwin,et al.  Refined structure of the nicotinic acetylcholine receptor at 4A resolution. , 2005, Journal of molecular biology.

[21]  A. Jabs,et al.  Non-proline cis peptide bonds in proteins. , 1999, Journal of molecular biology.

[22]  Niki M Zacharias,et al.  Cation-pi interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine. , 2002, Biochemistry.

[23]  H. Lester,et al.  In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. , 1998, Methods in enzymology.

[24]  J. Changeux,et al.  Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism. , 2005, Biophysical journal.

[25]  J Andrew McCammon,et al.  Agonist-mediated Conformational Changes in Acetylcholine-binding Protein Revealed by Simulation and Intrinsic Tryptophan Fluorescence* , 2005, Journal of Biological Chemistry.

[26]  J. Thornton,et al.  Influence of proline residues on protein conformation. , 1991, Journal of molecular biology.

[27]  Christophe Dugave,et al.  Cis-trans isomerization of organic molecules and biomolecules: implications and applications. , 2003, Chemical reviews.

[28]  P. Chau,et al.  Prediction of 5-HT3 receptor agonist-binding residues using homology modeling. , 2003, Biophysical journal.

[29]  Henry A. Lester,et al.  Cys-loop receptors: new twists and turns , 2004, Trends in Neurosciences.

[30]  M. Akabas,et al.  GABAA Receptor M2–M3 Loop Secondary Structure and Changes in Accessibility during Channel Gating* , 2002, The Journal of Biological Chemistry.

[31]  J. Trudell,et al.  Coupling of agonist binding to channel gating in the GABAA receptor , 2003, Nature.

[32]  T. Lectka,et al.  INTRAMOLECULAR CATALYSIS OF AMIDE ISOMERIZATION : KINETIC CONSEQUENCES OF THE 5-NH- -NA HYDROGEN BOND IN PROLYL PEPTIDES , 1998 .

[33]  C. Deane,et al.  The role and predicted propensity of conserved proline residues in the 5-HT3 receptor. , 2001, The Journal of biological chemistry.