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)

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Abstract

The phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP2 levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP2 concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP2 and yielded channels generating currents proportional to the PIP2 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 PIP2 by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding ∼150% of total microvillar phosphoinositides per second. PIP2 was resynthesized with a half-time of ∼50 s. When PIP2 resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of ∼40% loss of total PIP2 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. PIP2 levels were reduced ∼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.

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Hardie, R. C., Gu, Y., Martin, F., Sweeney, S. T., & Raghu, P. (2004). 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). Journal of Biological Chemistry, 279(46), 47773–47782. https://doi.org/10.1074/jbc.M407525200

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