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Polymerase chain reaction and rapid amplification of cDNA ends were used to isolate cDNAs encoding a 5-hydroxytryptamine 3 (5-HT 3 ) 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-HT 3 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-HT 3 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-HT 3 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-HT 3 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-HT 3 receptors, the guinea pig receptor showed prolonged desensitization kinetics. In addition, the guinea pig 5-HT 3 receptor did not respond to the selective 5-HT 3 receptor agonist PBG. Construction of chimeric receptors between guinea pig and human 5-HT 3 receptor sequences localized the differences in desensitization kinetics to the carboxyl-terminal domain and the ligand binding site to the aminoterminal domain of the receptor protein. Molecular determinants of the PBG binding site of the human 5-HT 3 receptor were localized to a 28-amino-acid spanning region adjacent to the M1 region.5-HT 3 Rs belong to the superfamily of ligand-gated ion channels that mediate fast synaptic transmission in the peripheral and central nervous systems (Peters et al., 1992;Yakel, 1992). These channels are composed of five identical or homologous subunits and their functional diversity generally is attributed to the presence of several different subunits that can coassemble to yield receptors with specific pharmacological and physiological properties (Betz, 1990). No such diversity has emerged for 5-HT 3 Rs. A single 5-HT 3 inotropic receptor subunit (5-HT 3 R-A) was cloned 6 years ago from the NCB20 neuroblastoma cell line (Maricq et al., 1991), but despite evidence for both pharmacological and biophysical variations between tissues and species, no further 5-HT 3 R subunits, like different ␣ or ␤ subunits, have been identified. 5-HT 3 R-A cDNA and a splice variant have been cloned from additional neuroblastoma cell lines and from mouse, rat, and human tissues. These subunits form functional homo-oligomeric 5-HT 3 Rs when expressed in oocytes or H...

Desensitization of ligand-gated ion channels shapes synaptic responses and provides critical neuroprotection at central synapses, yet the molecular mechanisms underlying the desensitization process are poorly understood. Using the whole-cell voltage-clamp technique, we investigated desensitization kinetics of recombinant human and guinea pig alpha-homomeric 5-hydroxytryptamine type 3 (5-HT(3A)) receptors heterologously expressed in human embryonic kidney 293 cells. Human 5-HT(3A) receptors desensitize 3.5 times faster than does the homologous receptor from guinea pigs. By constructing various chimeras and through site-directed mutagenesis, we have identified a single serine in the M1 region of the human 5-HT(3A) receptor sequence (S248) that, when substituted with threonine found in the equivalent guinea pig sequence (T254), conferred guinea pig-like kinetics on the time course of desensitization of the human receptor. Correspondingly, the reverse mutation (guinea pig T254S) resulted in a fast, human-like time constant of desensitization. Thus, the primary structure of the M1 region is an important determinant of desensitization kinetics of recombinant 5-HT(3A) receptors.

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