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 "100 nM", approximately 10-fold inhibition at <10nM or approximately 1 microM). However, experiments where the total BAPTA concentration was varied whilst free [Ca2+] was maintained constant indicated that inhibition of PLC at higher (>100 nM) nominal Ca2+ concentrations was independent of Ca2+ and due to inhibition by BAPTA itself (IC50 approximately 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC50 approximately 1mM). 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.
Hardie
Roger C
RC
Cambridge University, Department of Anatomy, Downing St, Cambridge CB2 3DY, UK. rch14@hermes.cam.ac.uk
D000818:Animals; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000187:drug effects / Q000201:enzymology*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D017956:Photoreceptor Cells, Invertebrate / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D029721:Drosophila Proteins; C053459:5,5'-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004533:Egtazic Acid; D010738:Type C Phospholipases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid