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- W2912668600 abstract "Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric proteins composed of subunits from CNGA3 and CNGB3 genes. Cone CNG channels transduce light information into electrical signals conducting both Na+ and Ca2+ ions. More than 100 mutations in the CNGA3 gene are associated with the inherited retinal disorder, achromatopsia 2 (ACHM2) which results in attenuation or loss of color vision. Classical techniques to measure CNG channel function use patch clamp electrophysiology providing high temporal and spatial resolution. Due to the low throughput of patch clamp electrophysiology, analysis of the functional properties of mutants by this method is challenging and time intensive. As a functional macroscopic screen, we developed a fluorescence-based, high-throughput Ca2+ flux assay of CNGA3 channels expressed in HEK293 cells. The 384-well microplate assay uses Rhod-4 AM to monitor Ca2+ influx and a yellow fluorescent tag to monitor cellular localization of the mutant protein subunits. 8-(4-chlorophenylthio)-cGMP (CPT-cGMP) activates channel openings in the assay. Here we demonstrate Ca2+ levels of wild-type CNGA3 channels along with three ACHM2-associated channel mutants, CNGA3-L633P, CNGA3-R427C, and CNGA3-T291R.The K0.5 values for CPT-cGMP activation in the Ca2+ flux assay were 9.4 μM for the wild-type CNGA3, 10.9 μM for the L633P mutant and 3.3 μM for the gain-of-function mutant R427C; the Nh values for each were ∼2.3. A CNGA3-T291R mutant had no activated flux activity. These values are in reasonable agreement with cGMP channel activation from patch data. The overall goal of developing this high throughput assay is to identify ACHM2-associated mutants that are misfolded. Candidates will be expressed with the CNGB3 subunit and screened for compounds that assist folding to recover channel function." @default.
- W2912668600 created "2019-02-21" @default.
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- W2912668600 date "2019-02-01" @default.
- W2912668600 modified "2023-09-26" @default.
- W2912668600 title "Development of a High-Throughput Ca2+ Flux Screening Assay to Monitor Cyclic Nucleotide-Gated Channel Activity and Evaluate Achromatopsia Disease Mutant Channel Function" @default.
- W2912668600 doi "https://doi.org/10.1016/j.bpj.2018.11.614" @default.
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