The Phosphorylation State of theDrosophilaTRP Channel Modulates the Frequency Response to Oscillating LightIn Vivo

Abstract

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.

Functional characterization of the three Drosophila retinal degeneration C (RDGC) protein phosphatase isoforms

“…Recently, we reported light- and RDGC-dependent dephosphorylation of TRP at Ser936 [ 8 ]. To investigate the general capacity of the rdgC ΔS , rdgC ΔSM , and rdgC ΔL flies to dephosphorylate pS936-TRP, we light and dark adapted flies for one hour and subjected fly heads to Western blot analyses ( Fig 4A ).…”

“…To investigate the general capacity of the rdgC ΔS , rdgC ΔSM , and rdgC ΔL flies to dephosphorylate pS936-TRP, we light and dark adapted flies for one hour and subjected fly heads to Western blot analyses ( Fig 4A ). Using a phosphospecific antibody that was raised against a phosphorylated synthetic peptide resembling TRP phosphorylated at S936 [ 8 – 10 ], we found that TRP was dephosphorylated in the light and phosphorylated in the dark at S936 in rdgC ΔS , rdgC ΔSM , and rdgC ΔL fly heads like in the wild type. Thus, RDGC-M and -L ( rdgC ΔS ), RDGC-L alone ( rdgC ΔSM ), as well as RDGC-S and -M ( rdgC ΔL ) are generally capable to mediate TRP dephosphorylation at S936 in the light.…”

“…RDGC contains an IQ-motif that binds to Ca 2+ /calmodulin and a mutation in this motif, rdgC I12E (but not rdgC I12A ), abolished calmodulin binding and resulted in retinal degeneration [ 7 ]. Besides dephosphorylation of rhodopsin, we recently showed that RDGC is also involved in the dephosphorylation of the TRP cation channel at Ser936 [ 8 ]. Interestingly, Ser936 is the only known TRP phosphorylation site that is phosphorylated in the dark and becomes dephosphorylated in the light [ 8 – 10 ], consistent with Ca 2+ -dependent activation of RDGC.…”

“…Besides dephosphorylation of rhodopsin, we recently showed that RDGC is also involved in the dephosphorylation of the TRP cation channel at Ser936 [ 8 ]. Interestingly, Ser936 is the only known TRP phosphorylation site that is phosphorylated in the dark and becomes dephosphorylated in the light [ 8 – 10 ], consistent with Ca 2+ -dependent activation of RDGC. As revealed by exchange of Ser936-TRP to Ala, preventing phosphorylation, or Asp, mimicking phosphorylation, dephosphorylation of TRP at Ser936 enables photoreceptor cells to process oscillating light stimuli of higher frequencies.…”

“…As revealed by exchange of Ser936-TRP to Ala, preventing phosphorylation, or Asp, mimicking phosphorylation, dephosphorylation of TRP at Ser936 enables photoreceptor cells to process oscillating light stimuli of higher frequencies. Thus, the phosphorylation state of TRP at Ser936 plays a role in the temporal aspect of light adaptation [ 8 ].…”