Light-dependent Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel

Abstract

Reversible protein phosphorylation is a well established mechanism for regulating the activity of ion channels. Typically, the pattern of phosphorylation of ion channels is complex, involving several phosphorylation sites with consensus sequences for a number of protein kinases, such as protein kinase C (PKC), 3 protein kinase A, calcium/calmodulin-dependent protein kinase, or casein kinase, which phosphorylate serine and threonine residues, as well as kinases phosphorylating tyrosine residues. For example, in the major delayed rectifier K ϩ channel Kv2.1, expressed in most central neurons, 16 phosphorylation sites have been identified by mass spectrometry, a subset of which contributes to graded modulation of voltagedependent gating (2).Transient receptor potential (TRP) channels constitute a protein family of about 30 unique homologs that are assigned to seven subfamilies on the basis of sequence homology: canonical TRPC, vanilloid TRPV, melastatin TRPM, polycystin TRPP, mucolipin TRPML, and ankyrin transmembrane proteins TRPA and NOMPC-like TRPN (3, 4). The founding member of this protein family is the Drosophila TRP channel, which, together with its homolog TRP-like (TRPL), is located in the rhabdomeral photoreceptor membrane of the fly compound eye and represents the major light-sensitive ion channel in this phospholipase C-mediated visual transduction cascade (5). Phosphorylation of several TRP channels has been described. Among the vertebrate TRPC channels, TRPC3 and TRPC6 are inhibited by phosphorylation events mediated by protein kinase C and protein kinase G (6 -8). In contrast, Src kinase activity is required for the activation of TRPC3 by diacylglycerol (9), and Fyn kinase phosphorylates and thereby increases the activity of TRPC6 (10). Abolition of the putative protein kinase C phosphorylation site Thr 635 in the S4/S5 linker region of TRPC3 by mutation results in increased channel activity and was found to underlie the phenotype of moonwalker mice, which is caused by loss of Purkinje cells (11). The regulation of the capsaicin-and heat-sensitive TRPV1 channel through phosphorylation of serine residues by protein kinase C is also well established (12)(13)(14). Phosphorylation of TRPV1 sensitizes this channel to capsaicin, heat, and other agonists. Besides protein kinase C, calcium/calmodulin-dependent kinase and protein kinase A were implicated in phosphorylation of TRPV1 (15, 16).The first TRP channel shown to become phosphorylated again was the Drosophila TRP channel. This channel is part of a signaling complex assembled by the INAD scaffold protein together with phospholipase C and an eye-enriched protein kinase C (eye-PKC) encoded by the inaC gene. It was shown initially for the larger fly Calliphora vicina and later also for Drosophila that the addition of ATP to the isolated signaling complex resulted in phosphorylation of TRP and INAD, suggesting that these two proteins of the signaling complex are targets of the associated protein kinase C (17)(18)(19)

Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity

“…A third potential role for phosphorylation of the MSL10 N-terminal domain is in the regulation of its ion channel properties, as has been shown for TPK1 (Latz et al, 2007), TRP (Voolstra et al, 2010), CLH-3b (Yamada et al, 2013), and PIP2 (Prado et al, 2013). Mutations outside the membrane-spanning pore of bacterial MscS homologs alter channel conductance Cox et al, 2013) and gating properties (Nomura et al, 2008;Vásquez et al, 2008;Belyy et al, 2010;Koprowski et al, 2011).…”

Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

“…Mass Spectrometry-Mass spectrometry and data analysis was performed as described before (10), except that a longer gradient (90 min) was used for peptide elution from the HPLC column, survey spectra were recorded in the Orbitrap detector in the m/z range of 250 to 2000, and data-dependent tandem mass spectra were generated for the seven most abundant peptide precursors in the linear ion trap. Mascot 2.3 (Matrix Science) and Sequest (Thermo Fisher Scientific) search results were loaded into Proteome Discoverer 1.3 (Thermo Fisher Scientific) for verification.…”

“…Gel Electrophoresis and Immunoblot Analysis-Protein extracts were generated as described (10). 4 l of SDS extraction buffer was used per fly head, and extraction at room temperature was extended to 1 h. Immunoblot analysis was carried out as described (10) using anti-TRPL (12) and anti-␤-tubulin antibodies (Developmental Studies Hybridoma Bank, University of Iowa).…”

“…Immunoprecipitation of TRPL Channels-Protein extracts of 80 fly heads per sample for immunoprecipitation with ␣-GFP or 300 fly heads per sample for immunoprecipitation with ␣-TRPL were obtained as described (10). 2 l of extraction buffer was used per fly head.…”

“…The founding member of the TRP protein family, the Drosophila TRP channel, together with its homolog TRPL (TRP-like) is expressed in photoreceptor cells of the fly compound eye and generates the receptor potential in the visual transduction cascade (7)(8)(9). A mass spectrometry analysis of posttranslational TRP modifications identified 21 phosphorylation sites (10). One of the light-dependent phosphorylation sites of TRP, Ser-982, was shown to be critical for the proper deactivation of the photoresponse (11), whereas the physiological function of the other phosphorylation sites is still elusive.…”