Isomerization of the proline in the M2-M3 linker is not required for activation of the human 5-HT3A receptor.
Each subunit of the cation-selective members of the Cys-loop family of ligand-gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5-hydroxytryptamine type 3A (5-HT(3)A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans-cis isomerization of the protein backbone at this position is integral to agonist-induced channel opening [Nature (2005) vol. 438, pp. 248-252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5-HT(3)A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist-mediated currents, compromise channel gating by 5-HT or inhibition of 5-HT-induced currents by either picrotoxin or d-tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca(2+) concentration and a 10-fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5-HT(3)A receptor function but indicate that trans-cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.
Paulsen
Isabelle M
IM
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Martin
Ian L
IL
Dunn
Susan M J
SM
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D005260:Female; D006639:Histidine / Q000235:genetics; D006801:Humans; D007536:Isomerism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000187:drug effects / Q000502:physiology; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology; D014364:Tryptophan / Q000235:genetics; D014982:Xenopus laevis
D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3; D012701:Serotonin; D006639:Histidine; D014364:Tryptophan; D011392:Proline