Evidence that 2-aminoethyl diphenylborate is a novel inhibitor of store-operated Ca²⁺ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors

The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P₃ receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P₃ receptors in the activation of store-operated Ca²⁺ channels (SOCs) [Ma. Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287. 1647 1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca²⁺ inflow (measured using fura-2) with no detectable effect on the release of Ca²⁺ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca²⁺ inflow was approx. 10μM. 2-APB also inhibited Ca²⁺inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca²⁺ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca²⁺ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca²⁺ after a vasopressin-induced plateau in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) had been established, the kinetics of the decrease in [Ca²⁺]_cyt were identical with those induced by the addition of 50μM Gd³⁺ (gadolinium). 2-APB did not inhibit the release of Ca²⁺ from intracellular stores induced by the addition of Ins(1,4,5)P₃ to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line. 2-APB inhibited thapsigargin-stimulated Ca²⁺ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P₃-induced inward current carried by Ca²⁺. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P₃ receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P₃ receptors in the activation of SOCs in other cell types is briefly discussed.