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 (R_max) 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: R_max mCPBG/R_max 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.

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