Molecular Cloning, Functional Expression, and Pharmacological Characterization of 5-Hydroxytryptamine₃ Receptor cDNA and Its Splice Variants from Guinea Pig

Abstract

Polymerase chain reaction and rapid amplification of cDNA ends were used to isolate cDNAs encoding a 5-hydroxytryptamine₃(5-HT₃) receptor subunit and its splice variants from guinea pig intestine. The amino acid sequence predicted from this cDNA is 81% homologous to the murine 5-HT₃ receptor subunits cloned from NCB20 and N1E-115 cells. The splice variants code for two proteins differing by a deletion of six amino acids located in the large intracellular loop between transmembrane domains M3 and M4. For characterization, the cloned 5-HT₃ cDNA was expressed in HEK 293 cells, and the electrophysiological and pharmacological properties of the recombinant ion/channel/receptor complex were investigated by patch clamping. Our data reveal that the cloned cDNAs code for guinea pig 5-HT₃ receptors, which functionally assemble as homo-oligomers. The kinetic behavior of the ion channel and its sensitivity to several agonists and antagonists were markedly different from those of the cloned 5-HT₃ receptors from mouse and human under similar experimental conditions. The agonists used were 5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, 1-phenylbiguanide (PBG), m-chlorophenylbiguanide, and the antagonists tropisetron and metoclopramide. In addition, 5-HT, PBG, and tropisetron were investigated through radioligand binding to isolated membranes. Compared with the human and murine 5-HT₃ receptors, the guinea pig receptor showed prolonged desensitization kinetics. In addition, the guinea pig 5-HT₃receptor did not respond to the selective 5-HT₃ receptor agonist PBG. Construction of chimeric receptors between guinea pig and human 5-HT₃ receptor sequences localized the differences in desensitization kinetics to the carboxyl-terminal domain and the ligand binding site to the amino-terminal domain of the receptor protein. Molecular determinants of the PBG binding site of the human 5-HT₃ receptor were localized to a 28-amino-acid spanning region adjacent to the M1 region.