A diacylglycerol-activated Ca2+ channel in PC12 cells (an adrenal chromaffin cell line) correlates with expression of the TRP-6 (transient receptor potential) protein.
The structures, and mechanisms of activation, of plasma membrane intracellular-messenger-activated, non-selective cation channels in animal cells are not well understood. The PC12 adrenal chromaffin cell line is a well-characterized example of a nerve cell. In PC12 cells, 1-oleolyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, initiated the inflow of Ca(2+), Mn(2+) and Sr(2+). Acetylcholine and thapsigargin initiated the inflow of Ca(2+) and Mn(2+), but not of Sr(2+). The activation of bivalent cation inflow by OAG: (i) was mimicked by another membrane-permeant diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, but not by the membrane-impermeant analogue 1-stearoyl-2-arachidonyl-sn-glycerol; (ii) was not blocked by staurosporin or chelerythrine, inhibitors of protein kinase C; (iii) was enhanced by RHC80267 and R50922, inhibitors of diacylglycerol lipase and diacylglycerol kinase respectively; and (iv) was inhibited by extracellular Ca(2+). When OAG was added after acetylcholine, the effect of OAG on Ca(2+) inflow was over-and-above that induced by acetylcholine. 2-Aminoethyl diphenylborate (2-APB) inhibited Ca(2+) inflow initiated by either acetylcholine or thapsigargin, but not that initiated by OAG. Flufenamic acid inhibited OAG-initiated, but not acetylcholine-initiated, Ca(2+) and Mn(2+) inflow. OAG-initiated Ca(2+) inflow was less sensitive to inhibition by SK&F96365 than acetylcholine-initiated Ca(2+) inflow. In polyadenylated RNA prepared from PC12 cells, mRNA encoding TRP (transient receptor potential) proteins 1-6 was detected by reverse transcriptase (RT)-PCR, and in lysates of PC12 cells the endogenous TRP-6 protein was detected by Western blot analysis. It is concluded that PC12 cells express a diacylglycerol-activated, non-selective cation channel. Expression of this channel function correlates with expression of the TRP-3 and TRP-6 proteins, which have been shown previously to be activated by diacylglycerol when expressed heterologously in animal cells [Hofmann, Obukhov, Schaefer, Harteneck, Gudermann, and Schultz (1999) Nature (London) 397, 259-263].
Tesfai
Y
Y
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, G.P.O. Box 2100, Adelaide, South Australia, 5001, Australia.
Brereton
H M
HM
Barritt
G J
GJ
D000109:Acetylcholine / Q000494:pharmacology; D000310:Adrenal Gland Neoplasms; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability; D019439:Chromaffin Cells / Q000166:cytology / Q000502:physiology; D004075:Diglycerides / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010446:Peptide Fragments / Q000276:immunology; D010673:Pheochromocytoma; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D013324:Strontium / Q000378:metabolism; D050052:TRPC Cation Channels; D014158:Transcription, Genetic / Q000187:drug effects
D015220:Calcium Channels; D004075:Diglycerides; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D012333:RNA, Messenger; D050052:TRPC Cation Channels; C044295:1,2-dioctanoylglycerol; D008345:Manganese; C075809:1-stearoyl-2-arachidonoylglycerol; C038237:1-oleoyl-2-acetylglycerol; D000109:Acetylcholine; D002118:Calcium; D013324:Strontium