title: Activation of heterologously expressed Drosophila TRPL channels: Ca2+ is not required and InsP3 is not sufficient.
abstract: Light-sensitive channels encoded by the Drosophila transient receptor potential-like gene (trpl) are activated in situ by an unknown mechanism requiring activation of Gq and phospholipase C (PLC). Recent studies have variously concluded that heterologously expressed TRPL channels are activated by direct Gq-protein interaction, InsP3 or Ca2+. In an attempt to resolve this confusion we have explored the mechanism of activation of TRPL channels co-expressed with a PLC-specific muscarinic receptor in a Drosophila cell line (S2 cells). Simultaneous whole-cell recordings and ratiometric Indo-1 Ca2+ measurements indicated that agonist (CCh)-induced activation of TRPL channels was not always associated with a rise in Ca2+. Internal perfusion with BAPTA (10 mM) reduced, but did not block, the response to agonist. In most cases, releasing caged Ca2+ facilitated the level of spontaneous channel activity, but similar concentrations (200-500 nM) could also inhibit TRPL activity. Releasing caged InsP3 invariably released Ca2+ from internal stores but had only a minor influence on TRPL activity and none at all when Ca2+ release was buffered with BAPTA. Caged InsP3 also failed to activate any light-sensitive channels in situ in Drosophila photoreceptors. Two phospholipase C inhibitors (U-73122 4 microM and bromo-phenacyl bromide 50 microM) reduced both spontaneous and agonist-induced TRPL activity in S2 cells. The results suggest that, as in situ, TRPL activation involves G-protein and PLC; that Ca2+ can both facilitate and in some cases inhibit TRPL channels, but that neither Ca2+ nor InsP3 is the primary activator of the channel.
authors.affiliation: Department of Anatomy, Cambridge University, UK. rch14@hermes.cam.ac.uk
authors.forename: R C
authors.initials: RC
authors.lastname: Hardie
authors.forename: P
authors.initials: P
authors.lastname: Raghu
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002148:Calmodulin-Binding Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050051:Transient Receptor Potential Channels; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D002148:Calmodulin-Binding Proteins; D002614:Chelating Agents; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D011994:Recombinant Proteins; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D010738:Type C Phospholipases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium