In vivo light-induced and basal phospholipase C activity in Drosophila photoreceptors measured with genetically targeted phosphatidylinositol 4,5-bisphosphate-sensitive ion channels (Kir2.1).
The phosphatidylinositol 4,5-bisphosphate (PIP(2))-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP(2) levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP(2) concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP(2) and yielded channels generating currents proportional to the PIP(2) levels relevant for phototransduction. To isolate Kir2.1 currents, recordings were made from mutants lacking both classes of light-sensitive transient receptor potential channels (TRP and TRPL). Light resulted in the effective depletion of PIP(2) by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding approximately 150% of total microvillar phosphoinositides per second. PIP(2) was resynthesized with a half-time of approximately 50 s. When PIP(2) resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of approximately 40% loss of total PIP(2) per minute. This loss was attributed primarily to basal PLC activity, because it was greatly decreased in norpA mutants lacking PLC. We tried to confirm this by using the PLC inhibitor U73122; however, this was found to act as a novel inhibitor of the Kir2.1 channel. PIP(2) levels were reduced approximately 5-fold in the diacylglycerol kinase mutant (rdgA), but basal PLC activity was still pronounced, consistent with the suggestion that raised diacylglycerol levels are responsible for the constitutive TRP channel activity characteristic of this mutant.
Hardie
Roger C
RC
Department of Anatomy, Cambridge University, Downing St., Cambridge CB2 3DY, United Kingdom. rch14@hermes.cam.ac.uk
Gu
Yuchun
Y
Martin
Fernando
F
Sweeney
Sean T
ST
Raghu
Padinjat
P
D000818:Animals; D004331:Drosophila melanogaster / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D018390:Gene Targeting; D008027:Light; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D017956:Photoreceptor Cells, Invertebrate / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D017354:Point Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
C489461:Kir2.1 channel; D019269:Phosphatidylinositol 4,5-Diphosphate; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases