© 2006 The Japanese Biochemical Society.
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Muscarinic Acetylcholine Receptors Activate TRPC6 Channels in PC12D Cells via Ca2+ Store–Independent Mechanisms
1 Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033; and 2 Graduate Program in Molecular, Cell and Developmental Biology, and 3 Departments of Pharmacology and 4 Psychiatry, College of Medicine and Public Health, The Ohio State University, 333 West 10th Ave., Columbus, OH 43210, USA
To whom correspondence should be addressed at: Department of Pharmacology, College of Medicine and Public Health, 5072C Graves Hall, 333 West 10th Ave, Columbus, Ohio. Tel: +1-614-688-4573, Fax: +1-614-292-7232, E-mail: saffen.1{at}osu.edu
In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6.
* Present address: Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church St. S.E., Minneapolis, MN 55455, USA.
Present address: Department of Biology, Pennsylvania State University, 113 Life Science Bldg, University Park, PA 16801, USA.
Present address: Department of Neuroscience/Institute for Cell Engineering, The Johns Hopkins University, BRB 729, 733 N. Broadway, Baltimore, MD 21205, USA.
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