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)
photoreceptors respond to oscillating light of high frequency (∼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic ( ) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid ( ) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted mutant flies showed relatively high S936-TRP phosphorylation levels and light-dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions. photoreceptors exhibit high temporal resolution as manifested in frequency response to oscillating light of high frequency (≤∼100 Hz). Light rearing conditions modulate the maximal frequency detected by photoreceptors, thus enabling them to maintain high sensitivity to light and high temporal resolution. However, the precise mechanisms for this process are not fully understood. Here, we show by combination of biochemistry and electrophysiology that transient receptor potential (TRP) channel dephosphorylation at a specific site is a fast, light-activated and Ca-dependent regulatory process. TRP dephosphorylation affects the detection limit of oscillating light according to the adaptation state of the photoreceptor cells by shifting the detection limit to higher frequencies upon light adaptation. This novel mechanism thus adjusts dynamic processing of visual information under varying light conditions.
Signaling at the plasma membrane is modulated by up- and downregulation of signaling proteins. A prominent example for this type of regulation is the Drosophila TRPL ion channel that changes its spatial distribution within the photoreceptor cell. In dark-raised flies TRPL is localized in the rhabdomeral photoreceptor membrane and it translocates to the cell body upon illumination. It has been shown that TRPL translocation depends on the activation of the phototransduction cascade and requires the presence of functional rhodopsin as well as Ca2+-influx through a second lightactivated ion channel, TRP. However, little is known about the cell biological mechanism underlying TRPL translocation. Here we describe a FRT/FLP screen designed to isolate mutants defective in TRPL internalization based on the localization of eGFP-tagged TRPL in the eyes of living flies. We mutated chromosome arms 2L, 2R and 3R and isolated 12 mutants that failed to internalize TRPL. We found that four mutants did not complement genes known to affect TRPL translocation, which are trp, ninaE and inaD. Two of the isolated mutants represent new alleles of trp and ninaE. The trp allele contains a premature stop codon after amino acid 884, whereas the ninaE allele has a mutation resulting in the substitution P193S. As determined biochemically no TRP or rhodopsin protein, respectively, was expressed in the eyes of these mutants. The absence of TRP or rhodopsin in the isolated mutants readily explains the defect in TRPL internalization and proves the feasibility of our genetic screen.
Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation.
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