Phosphorylation of the transient receptor potential (TRP) ion channel in Drosophila vision

• Applicant: Dr. Olaf Voolstra
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2010 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 190739464

The opening of the transient receptor potential (TRP) ion channel is the last step of the phototransduction cascade in Drosophila. In previous work, we identified 28 TRP phosphorylation sites, 27 of which are located within the intracellular C-terminus. A subset of the C-terminal phosphorylation sites, including T849, displays enhanced phosphorylation in the light, whereas a single site, S936, exhibits enhanced phosphorylation in the dark. We found eye-specific protein kinase C (eye-PKC) to be involved in phosphorylation of T849 and retinal degeneration C (RDGC) to be involved in dephosphorylation of S936. Mutation of T849 to A eliminating phosphorylation and mutation of S936 to D mimicking phosphorylation both resulted in prolonged deactivation of the photoresponse in vivo. Preliminary data point to an involvement of S936 phosphorylation in light adaption in Drosophila.The proposed project aims at elucidating the role of TRP phosphorylation at T849 by eye-PKC and dephosphorylation at S936 by RDGC. We will generate a transgenic fly expressing a modified TRP channel that harbors both the T849A and the S936D exchanges to investigate a possible synergistic regulation of response deactivation by both sites. To investigate the global role of TRP phosphorylation, we will also generate a fly that expresses a TRP channel in which all phosphorylation sites will be eliminated by mutation to A. We will also revert the amino acids 849 and 936 to T and S, respectively, to investigate a possible rescue of phenotypes.A prerequisite of vertebrate PKC activation is the phosphorylation by PKC kinase and the concomitant autophosphorylation at two additional sites. These three phosphorylation sites are conserved in Drosophila eye-PKC. We observed phosphorylation of the first autophosphorylation site by mass spectrometry and now intend to investigate whether the two other sites are phosphorylated in Drosophila eye-PKC as well. We will generate transgenic flies that express eye-PKCs with modified phosphorylation sites and will investigate the physiological consequences of modification of phosphorylation sites in these flies. Additionally, we will search for new eye-PKC target proteins in a shotgun approach by comparing phosphopeptide abundancies in eye-PKC null mutant flies and wild type flies.RDGC was shown to dephosphorylate rhodopsin at elusive C-terminal sites. We will identify rhodopsin phosphorylation sites by mass spectrometry. rdgC null mutants exhibit light-induced retinal degeneration and prolonged deactivation of the light response. By expressing a C-terminally truncated rhodopsin lacking the phos-phorylation sites, and by expressing trpS936A in an rdgC null mutant background we will dissect the influences of TRP and rhodopsin hyper¬phos¬phory¬lation in an rdgC null mutant.The proposed project will help to understand the interplay of kinases and phosphatases in the regulation of ion channels.

• DFG Programme: Research Grants