Muscarinic acetylcholine receptors activate TRPC6 channels in PC12D cells via Ca2+ store-independent mechanisms.
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.
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033.
Lei
L
Zhang
Feifan
F
Guo
Ju Young
JY
Kim
David
D
Saffen
D000818:Animals; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects*; D016716:PC12 Cells; D051381:Rats; D011976:Receptors, Muscarinic / Q000502:physiology*; D050052:TRPC Cation Channels / Q000378:metabolism*
D018679:Cholinergic Agonists; D011976:Receptors, Muscarinic; D050052:TRPC Cation Channels; C492145:Trpc6 protein, rat; D001464:Barium; D002217:Carbachol; D002118:Calcium