Browsing by Subject "Eye"

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Untersuchung der lichtabhängigen Phosphorylierung des TRP-Kanals von Drosophila melanogaster
(2015) Bartels, Jonas-Peter; Huber, Armin
The phototransduction cascade in the eye of Drosophila melanogaster culminates in the opening of the ion channels TRP and TRPL. The hereby increased intracellular concentration of sodium and calcium ions underlies the formation of the photoreceptor potential. The TRP channel is subject to light-dependent phosphorylation. 15 of its phosphorylation sites exhibit increased phosphorylation upon illumination and one phosphorylation site exhibits increased phosphorylation in the dark. When this work was started, neither the function of light-dependent TRP phosphorylation nor the involved kinases and phosphatases were known. Therefore, in the present work, kinases and phosphatases that affect the phosphorylation pattern of the TRP channel were identified. Towards this end a candidate screen of 79 kinase and phosphatase mutants was performed using three different phosphospecific antibodies. In this screen, eight kinases and one phosphatase were identified that affect the phosphorylation of one or more of the three tested phosphorylation sites. Eye-specific protein kinase C (ePKC) and protein kinase C 53E (PKC53E) mutants exhibited reduced phosphorylation at T849 of the TRP channel, while mutants of the Rolled kinase and the alpha-subunit of AMP-dependent kinase showed an increased phosphorylation at this site. The mutants of Casein kinase Ialpha, Licorne, Tao, and Metallophosphoesterase (MPPE) exhibited reduced phosphorylation of the site T864. The retinal degeneration C (RDGC) phosphatase null mutant demonstrated a dramatically increased phosphorylation at the phosphorylation site S936. From these identified kinase and phosphatase mutants, only the mutants of the kinase ePKC and of the phosphatase RDGC displayed physiological abnormalities in terms of an already described slow deactivation of the light response in ERG measurements. To answer the question whether the altered phosphorylation of the TRP ion channel in the ePKC and RDGC mutants underlies the prolonged deactivation of the light response, transgenic flies were generated that express modified TRP channels. To this end, the ePKC-dependent TRP phosphorylation site T849 or the RDGC-dependent phosphorylation site S936 of the TRP channel were replaced by the amino acid alanine or aspartic acid. The biochemical analysis of these four transgenic flies revealed wild type characteristics of the modified channels with respect to subcellular localization, the interaction with the scaffold protein INAD, multimerization, and the expression rate. However, electrophysiological studies of these transgenic flies revealed a prolonged deactivation time when T849 was exchanged to alanine and in addition S936 was exchanged to aspartic acid. Whereas the exchange of a serine or threonine to alanine prevents phosphorylation, the exchange to aspartic acid typically mimics phosphorylation of this site. In the wild type situation, T849 is phosphorylated in the light and becomes dephosphorylated in the dark whereas for S936 the exact opposite is the case. The amino acid exchanges at the two phosphorylation sites thus mimic the phosphorylation pattern of dark-adapted wild type flies. In summary, the results demonstrate the involvement of eight kinases and one phosphatase in generating of the phosphorylation pattern of the TRP ion channel. Two of the phosphorylation sites of the TRP ion channel mediate a rapid deactivation of the light response.