Inhibition of phospholipase C activity in Drosophila photoreceptors by 1,2-bis(2-aminophenoxy) ethane N,N,N′,N′-tetraacetic acid (BAPTA) and di-bromo BAPTA

In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP2. In cells loaded via patch pipettes with varying concentrations of Ca2+ buffered by 4 mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca2+ (maximum at 100nM, similar to 10-fold inhibition at < 10 nM or similar to 1 mu M). However, experiments where the total BAPTA concentration was varied whilst free [Ca2+] was maintained constant indicated that inhibition of PLC at higher (> 100nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 similar to 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 similar to 1 mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca2+ chelators, whilst non-physiologically low (< 100 nM) levels of Ca2+ suppress both basal and light-induced PLC activity. (c) 2005 Elsevier Ltd. All rights reserved.