Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein.
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.
Boulay
G
G
Department of Anesthesiology, University of California, Los Angeles, California 90095-1778, USA.
Zhu
X
X
Peyton
M
M
Jiang
M
M
Hurst
R
R
Stefani
E
E
Birnbaumer
L
L
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D050052:TRPC Cation Channels; D014162:Transfection
D015220:Calcium Channels; D018076:DNA, Complementary; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C489571:TRPC4 ion channel; C096351:transient receptor potential cation channel, subfamily C, member 1; D008345:Manganese; D002217:Carbachol; D019204:GTP-Binding Proteins; D002118:Calcium