title: 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.
abstract: The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P(3) receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P(3) 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. 10 microM. 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 50 microM Gd(3+) (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P(3) 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)P(3)-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)P(3) receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P(3) receptors in the activation of SOCs in other cell types is briefly discussed.
authors.affiliation: Department of Medical Biochemistry, School of Medicine, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia.
authors.forename: R B
authors.initials: RB
authors.lastname: Gregory
authors.forename: G
authors.initials: G
authors.lastname: Rychkov
authors.forename: G J
authors.initials: GJ
authors.lastname: Barritt
mesh_terms: D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004347:Drug Interactions; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D001896:Boron Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D014667:Vasopressins; D019284:Thapsigargin; C109986:2-aminoethoxydiphenyl borate; D002118:Calcium; D016257:Fura-2