Activation of TRP Channels by Phosphoinositide Depletion and Protons in Drosophila Photoreceptors

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Phototransduction in Drosophila microvillar photoreceptors is mediated via a G-protein coupled phospholipase C (PLCβ4 encoded by the norpA gene), resulting in the activation of two classes of Ca2+ and cation permeable channels, TRP and TRPL. Representative of the TRPC subfamily, these are the founding members of the TRP ion channel superfamily, but exactly how the channels are activated is unresolved. PLC's obvious action is to hydrolyze PIP2 generating DAG and InsP3 whilst simultaneously reducing PIP2, the concentration of which can be accurately controlled using genetically encoded PIP2 biosensors to monitor levels. The fact that hydrolysis of PIP2 by PLC also releases a proton is seldom recognized and has neither been measured in vivo, nor implicated previously in a signaling context. We have now shown that light induces a rapid (< 10 ms) acidification originating in the microvilli which is eliminated in norpA mutants lacking PLC. Furthermore, following depletion of PIP2 and other phosphoinositides by a variety of experimental manipulations, both TRP and TRPL channels become remarkably sensitive to rapid and reversible activation by lipophilic protonophores such as 2-4 dinitrophenol, whilst heterologously expressed TRPL channels can be directly activated by acidification of the cytosolic surface of inside-out patches. These results indicate that a combination of phosphoinositide depletion and cytosolic acidification is sufficient to activate the light-sensitive channels. Together with the demonstration of light-induced, PLC dependent acidification, this suggests that TRPC channels in Drosophila photoreceptors may be activated in a combinatorial fashion by PLC's dual action of phosphoinositide depletion and proton release.