A diacylglycerol-activated Ca2+ channel in PC12 cells (an adrenal chromaffin cell line) correlates with expression of the TRP-6 (transient receptor potential) protein.

摘要

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].