InsP(3)-induced Ca2+ release in permeabilized invertebrate photoreceptors:a link between phototransduction and Ca2+ stores

Using the low-affinity fluorescent Ca2+ indicators, Mag-Fura-2 and Mag-Furs Red, we studied light- and InsP(3)-induced Ca2+ release in permeabilized m icrovillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two m ajor components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriat ely localized in photoreceptors. Whereas phospholipase-C was abudantly expr essed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the periphe ral plasma membrane with saponin allowed direct measurements of luminal fre e Ca2+ concentration (Ca-L) changes. Confocal Ca2+ imaging using Mag-Fura R ed demonstrated that Ins(1,4,5)P-3 mobilizes Ca2+ from SER. As detected wit h Mag-Fura-2, a brief 50 ms light flash activated rapid Ca2+ depletion of S ER, followed by an effective refilling within 1 min of dark adaptation afte r the light flash. Sensitivity to Ins(1,4,5)P-3 of the Ca2+ release from SE R in leech photoreceptors was accompanied by irreversible uncoupling of pho totransduction from Ca2+ release. Depletion of Ca2+ stores was induced by I ns(1,4,5)P-3 (EC50=4.75 muM) and the hyper-potent agonist adenophostin A (E C50=40 nM) while the stereoisomer L-myo Ins(1,4,5)P-3 was totally inactive, Ins(1,4,5)P-3- or adenophostin A-induced Ca2+ release was inhibited by 0.1 -1 mg/ml heparin. The Ca2+ pump inhibitors, cyclopiazonic acid and thapsiga rgin, in the presence of Ins(1,4,5)P-3, completely depleted Ca2+ stores in leech photoreceptors. (C) 2001 Harcourt Publishers Ltd.