Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion

Abstract

The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro-Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+o-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trpl cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2–17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5´-[γ-thio]triphosphate (GTP[S]), guanosine 5´-[β,γ-imido]triphosphate (p[NH]ppG) and AlF4-, but not guanosine 5´-[β-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] or AlF4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.