Regulation of the TRP calcium channel by eye-PKC in Drosophila

Abstract

The transient receptor potential (TRP) ion channels are implicated in diverse physiological processes, however their regulatory mechanisms remain obscure. In Drosophila, TRP is a calcium channel mediating the light-dependent depolarization of photoreceptor cells. TRP associates with inactivation-no-afterpotential D (INAD), a scaffolding protein that forms a macromolecular complex by also tethering a phospholipase Cbeta, NORPA, and an eye-specific protein kinase C, eye-PKC. It is well established that eye-PKC regulates deactivation of the visual response. Moreover, deactivation is regulated by the interaction between INAD and TRP, as abrogation of this interaction in InaDp215 results in slow deactivation similar to that of inaCp209 lacking eye-PKC. Eye-PKC was shown previously to phosphorylate TRP in vitro. In this thesis we investigated the mechanism by which eye-PKC regulates TRP to achieve fast deactivation of the visual signaling. We identified Ser982 of TRP as an eye-PKC phosphorylation site by an in vitro kinase assay. We show that phosphorylation of TRP by eye-PKC is dependent on both eye-PKC and INAD, suggesting that INAD acts to anchor eye-PKC to TRP. Importantly, we show by mass spectrometry that TRP is indeed phosphorylated by eye-PKC in vivo: phosphorylated peptides spanning Ser982 were observed in TRP isolated from light adapted wild-type but not from inaCp209 flies. To gain insight into the functional significance of phosphorylation at Ser982 of TRP, we generated transgenic flies expressing a modified TRP containing an Ala substitution at Ser982. We demonstrated that transgenic flies exhibited abnormal deactivation, indicating that phosphorylation of TRP at Ser982 by eye-PKC is critical for deactivation of the visual signaling. Interestingly, the slow deactivation defect in the transgenic flies is similar to that of InaDp215 in which TRP fails to associate with INAD, suggesting that the deactivation defect of InaDp215 is due to a loss of TRP phosphorylation by eye-PKC. Taken together, these findings support the notion that the INAD macromolecular complex is important for termination of the visual response, as it positions eye-PKC in close proximity to TRP to promote fast inactivation of the TRP channel.