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.

No abstract

SummaryFamily members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-induced current. The formation of functional TRP-TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP-TRPL channels using coimmunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and is being evaluated. The development of an efficient downstream purification process (DSP) enables the vaccine to be advanced to clinical trials. The DSP must eliminate impurities, either process- or product-related, to yield a sufficient product with high purity, potency and quality. To acquire critical information on process restrictions and qualities, the application of in-line monitoring is vital and should significantly impact the process yield, product quality and economy of the entire process. Here, we describe an in-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining the concentrations of metabolites, nutrients and a host cell protein using the automatic chemistry analyzer, Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adopting this technique during the purification process improves the process yield and the product quality and enhances the economy of the entire downstream process for biotechnology and bio pharmaceutical products.

Drosophila photoreceptors respond to oscillating light of high frequency (∼100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca-dependent process. We further found that this process affects the detection limit of high frequency oscillating light. Accordingly, transgenic Drosophila, which do not undergo phosphorylation at the S936-TRP site (trp), revealed a short time-interval before following the high stimulus frequency (oscillation-lock response) in both dark- and light-adapted flies. In contrast, the trp transgenic flies, which mimic constant phosphorylation, showed a long-time interval to oscillation-lock response in both dark- and light-adapted flies. Here we extend these findings by showing that dark-adapted trp flies reveal light-induced current (LIC) with short latency relative to trp or trp flies, indicating that the channels are a limiting factor of response kinetics. The results indicate that properties of the light-activated channels together with the dynamic light-dependent process of TRP phosphorylation at the S936 site determine response kinetics.