Arginine 222 in the pre-transmembrane domain 1 of 5-HT3A receptors links agonist binding to channel gating.
Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3A receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3A receptors.
Hu
Xiang-Qun
XQ
Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism/NIH, Park Building Room 150, Bethesda, MD 20892-8115, USA. xhu@mail.nih.gov
Zhang
Li
L
Stewart
Randall R
RR
Weight
Forrest F
FF
D000595:Amino Acid Sequence; D000818:Animals; D001058:Apomorphine / Q000494:pharmacology; D001120:Arginine; D002460:Cell Line; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000502:physiology; D012701:Serotonin / Q000031:analogs & derivatives / Q000494:pharmacology; D058827:Serotonin 5-HT3 Receptor Agonists; D014162:Transfection
D044406:Receptors, Serotonin, 5-HT3; D058827:Serotonin 5-HT3 Receptor Agonists; D012701:Serotonin; D001120:Arginine; D001058:Apomorphine