Mechanism of inhibition of TRPC cation channels by 2-aminoethoxydiphenylborane

We investigated the actions of the organoborane, 2-amino-ethoxydiphenylborane (2APB), on Ca2+ signaling in wild-type human embryonic kidney (HEK) 293 cells and in HEK293 cells stably expressing canonical transient receptor potential (TRPC) channels. Previous reports have suggested that 2APB inhibits agonist activation of TRPC channels because of its ability to act as a membrane-permeant inhibitor of inositol 1,4,5-trisphosphate (IP3) receptors. 2APB was specifically said to inhibit TRPC3 channels when activated through a phospholipase Clinked receptor but not when activated more directly by a synthetic diacylglycerol, oleyl-acetyl-glycerol (OAG) [Science (Wash DC) 287: 1647-1651, 2000]. However, we subsequently reported that IP3 does not activate TRPC3; rather the mechanism of activation by phospholipase C-linked receptors seemed to result from diacylglycerol [J Biol Chem 278: 16244-16252, 2003]. Thus, the current study was carried out to address the mechanism of action of 2APB in inhibiting TRPC channels. We found that, although the release of Ca2+ by a muscarinic agonist was reduced by high concentrations of 2APB, this effect was indistinguishable from that seen when stores were discharged by thapsigargin, which does not involve IP3 receptors. This indicates that 2APB is incapable of significant inhibition of IP3 receptors when applied to intact cells. We found that 2APB partially inhibits divalent cation entry in cells expressing TRPC3, TRPC6, or TRPC7 and that this partial inhibition was observed whether the channels were activated by a muscarinic agonist or by OAG. Thus, as concluded for store-operated channels, 2APB seems to inhibit TRPC channels by a direct mechanism not involving IP3 receptors.