Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization.
In this study, we report the molecular cloning of cDNAs encoding three distinct isoforms of rat (r) TRP6 Ca(2+) channels. The longest isoform, rTRP6A, contains 930 amino acid residues; rTRP6B lacks 54 amino acids (3-56) at the N terminus, and rTRP6C is missing an additional 68 amino acids near the C terminus. Transient transfection of COS cells with expression vectors encoding rTRP6A or rTRP6B increased Ca(2+) influx and gave rise to a novel Ba(2+) influx after activation of M(5) muscarinic acetylcholine receptors. By contrast, passive depletion of intracellular Ca(2+) stores with thapsigargin did not induce Ba(2+) influx in cells expressing rTRP6 isoforms. Ba(2+) influx was also stimulated in rTRP6A-expressing cells after exposure to the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), but rTRP6B-expressing cells failed to show OAG-induced Ba(2+) influx. Expression of a rTRP6 N-terminal fragment of rTRP6B or rTRP6A antisense RNA blocked M(5) muscarinic acetylcholine receptor-dependent Ba(2+) influx in COS cells that were transfected with rTRP6 cDNAs. Together these results suggest that rTRP6 participates in the formation of Ca(2+) channels that are regulated by a G-protein-coupled receptor, but not by intracellular Ca(2+) stores. In contrast to the results we obtained with rTRP6A and rTRP6B, cells expressing rTRP6C showed no increased Ca(2+) or Ba(2+) influxes after stimulation with carbachol and also did not show OAG-induced Ba(2+) influx. Glycosylation analysis indicated that rTRP6A and rTRP6B are glycosylated in COS cells, but that rTRP6C is mostly not glycosylated. Together these results suggest that the N terminus (3-56 amino acids) is crucial for the activation of rTRP6A by diacylglycerol and that the 735-802 amino acid segment located just downstream from the 6th transmembrane segment may be required for processing of the rTRP6 protein.
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. zhanglei@m.u-tokyo.ac.jp
L
L
Zhang
D
D
Saffen
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000493:pharmacokinetics; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004075:Diglycerides / Q000494:pharmacology; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D016372:RNA, Antisense / Q000494:pharmacology; D051381:Rats; D043642:Receptor, Muscarinic M5; D011976:Receptors, Muscarinic / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050052:TRPC Cation Channels; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D015220:Calcium Channels; D004075:Diglycerides; D020033:Protein Isoforms; D016372:RNA, Antisense; D043642:Receptor, Muscarinic M5; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D001464:Barium; D019284:Thapsigargin; C038237:1-oleoyl-2-acetylglycerol; D002217:Carbachol; D002118:Calcium