Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+.
The TM2-TM3 extracellular loop is critical for activation of the Cys-loop family of ligand-gated ion channels. The contribution of aspartate 298 (D298), an amino acid that links the transmembrane domain 2 (TM2) to the TM2-TM3 loop, in mouse 5-hydroxytryptamine(3A) (5-HT(3A)) receptor function was probed with site-directed mutagenesis in the present study. This negatively charged residue was replaced with an alanine to neutralize the charge, with a glutamate to conserve the charge, or with an arginine to reverse the charge. Human embryonic kidney 293 (HEK 293) cells transfected with the wild-type and mutant receptors were studied by combining whole-cell patch-clamp recording with fast agonist application. The D-->A or D-->R mutations resulted in a receptor with reduced 5-HT potency, and accelerated kinetics of desensitization and deactivation. In addition, the efficacy of partial agonists was reduced by the D-->A mutation. The D-->E mutation produced a receptor with properties similar to those of the wild-type receptor. In addition, the potential role of this residue in modulation of the receptor by extracellular calcium ([Ca(2)(+)](o)) was investigated. Increasing [Ca(2)(+)](o) inhibited 5-HT-activated currents and altered receptor kinetics in a similar manner in the wild-type and D298E receptors, and this alteration was eliminated by the D-->A and D-->R mutations. Our data suggest that the charge at D298 participates in transitions between functional states of the 5-HT(3A) receptor, and provide evidence that the charge of the side-chain at residue D298 contributes to channel gating kinetics and is crucial for Ca(2)(+) modulation.
Hu
Xiang-Qun
XQ
Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20852, USA.
Lovinger
David M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004298:Dopamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000031:analogs & derivatives / Q000494:pharmacology; D058827:Serotonin 5-HT3 Receptor Agonists; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014162:Transfection
D044406:Receptors, Serotonin, 5-HT3; D058827:Serotonin 5-HT3 Receptor Agonists; D017366:Serotonin Receptor Agonists; D001224:Aspartic Acid; D012701:Serotonin; C045918:2-methyl-5-HT; D002118:Calcium; D004298:Dopamine