Cloning and Expression of a Novel Mammalian Homolog ofDrosophila Transient Receptor Potential (Trp) Involved in Calcium Entry Secondary to Activation of Receptors Coupled by the Gq Class of G Protein

Guylain Boulay,Xi Zhu,Mike Peyton,Meisheng Jiang,Raymond Hurst,Enrico Stefani,Lutz Birnbaumer

doi:10.1074/jbc.272.47.29672

Abstract

Hormonal stimulation of Gq-protein coupled receptors triggers Ca2+ mobilization from internal stores. This is followed by a Ca2+ entry through the plasma membrane. Drosophila Trp and Trpl proteins have been implicated in Ca2+ entry and three mammalian homologues of Drosophila Trp/Trpl, hTrp1, hTrp3 and bTrp4 (also bCCE) have been cloned and expressed. Using mouse brain RNA as template, we report here the polymerase chain reaction-based cloning and functional expression of a novel Trp, mTrp6. The cDNA encodes a protein of 930 amino acids, the sequence of which is 36.8, 36.3, 43.1, 38.6, and 74. 1% identical to Drosophila Trp and Trpl, bovine Trp4, and human Trp1 and Trp3, respectively. Transient expression of mTrp6 in COS.M6 cells by transfection of the full-length mTrp6 cDNA increases Ca2+ entry induced by stimulation of co-transfected M5 muscarinic acetylcholine receptor with carbachol (CCh), as seen by dual wavelength fura 2 fluorescence ratio measurements. The mTrp6-mediated increase in Ca2+ entry activity was blocked by SKF-96365 and La3+. Ca2+ entry activity induced by thapsigargin was similar in COS cells transfected with or without the mTrp6 cDNA. The thapsigargin-stimulated Ca2+ entry could not be further stimulated by CCh in control cells but was markedly increased in mTrp6-transfected cells. Records of whole cell transmembrane currents developed in response to voltage ramps from -80 to +40 mV in control HEK cells and HEK cells stably expressing mTrp6 revealed the presence of a muscarinic receptor responsive non-selective cation conductance in Trp6 cells that was absent in control cells. Our data support the hypothesis that mTrp6 encodes an ion channel subunit that mediates Ca2+ entry stimulated by a G-protein coupled receptor, but not Ca2+ entry stimulated by intracellular Ca2+ store depletion.

Highlights

In many cell types, activation of a Gq-protein coupled receptor leads to the production of inositol 1,4,5-trisphosphate (IP3)1
As dTrp and Drosophila Trp-like (dTrpl) appeared to be related to some mammalian ion channels and are involved in Ca2ϩ entry upon stimulation of a phosphoinositide mobilizing receptor and Ca2ϩ store depletion, it was suggested that a vertebrate homologue of Trp should exist and that this homologue could be involved in vertebrate Ca2ϩ entry [17, 27, 28]
Direct participation of a Trp protein in mediating Ca2ϩ entry was inferred from the finding that the combined expression of partial antisense cDNAs of the six murine Trps led to loss of agonist activated Ca2ϩ entry [32]

Summary

Introduction

Activation of a Gq-protein coupled receptor leads to the production of inositol 1,4,5-trisphosphate (IP3)1. Addition of 20 ␮M CCh after the TG-induced Ca2ϩ entry had reached a plateau, produced a marked further increase in [Ca2ϩ]i in mTrp6-transfected cells but not in control cells.

Results

Conclusion