title: Different efficacy of specific agonists at 5-HT3 receptor splice variants: the role of the extra six amino acid segment.
abstract: 1. Whole cell voltage clamp electrophysiology and radioligand binding were used to examine the agonist characteristics of the two splice variants of the 5-HT3 receptor which have been cloned from neuronal cell lines. Homo-oligomeric 5-HT3 receptors were examined in HEK 293 cells stably transfected with either long (5-HT3-L) or short (5-HT3-S) receptor subunit DNAs. 2. Functional homo-oligomeric receptors were formed from both subunits, and responses to 5-HT3 receptor agonists (5-hydroxytryptamine (5-HT), 2-methyl 5-HT and m-chlorophenylbiguanide) were qualitatively similar. 3. Maximum currents (Rmax) elicited by the 5-HT3 receptor agonists m-chlorophenylbiguanide (mCPBG) and 2-methyl-5-HT (2-Me-5-HT), as compared to 5-HT, differed in the two splice variants: Rmax mCPBG/Rmax 5-HT values were 0.68+/-0.04 and 0.91+/-0.01 in 5-HT3-L and 5-HT3-S receptors, respectively. Comparable values for 2-Me-5-HT were 0.30+/-0.02 and 0.23+/-0.02. 4. Radioligand binding data showed no difference in affinity of agonist or antagonist binding sites; thus the six amino acid deletion appears to cause differences in agonist efficacy. 5. The role of the 6 amino acid insertion was further investigated by use of site-directed mutagenesis to create two mutant receptors, one where serine 286 was replaced with alanine, and the second where all 6 amino acids were replaced with alanines. 6. Examination of the mutant receptors when stably expressed in HEK 293 cells revealed agonist properties resembling long and not short 5-HT3 receptors. Thus specific amino acids in this region are not responsible for the observed differences. 7. The data show intracellular structure can have significant effects on ligand-gated ion channel function, and suggest that minor changes in structure may be responsible for differences in function observed when ligand-gated ion channel proteins are modulated intracellularly.
authors.affiliation: Departmento de Ciencias Medicas, Universidad de Santiago de Chile.
authors.forename: M I
authors.initials: MI
authors.lastname: Niemeyer
authors.forename: S C
authors.initials: SC
authors.lastname: Lummis
mesh_terms: D001645:Biguanides / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D016297:Mutagenesis, Site-Directed; D012326:RNA Splicing; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3; D012701:Serotonin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000378:metabolism*
chemical_list: D001645:Biguanides; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D017366:Serotonin Receptor Agonists; D012701:Serotonin; C045918:2-methyl-5-HT; C065734:1-(3-chlorophenyl)biguanide