Affiliation:
1. Jilin Medical University
Abstract
Abstract
Transient receptor potential cation channel subfamily V (TRPV4) is a widely expressed multimodal gated ion channel that transports Ca2+ intracellularly upon opening and plays an important role in many physiological and pathological processes. However, existing TRPV4 channel regulators lack specificity and are ineffective, and available screening methods are not suitable for high-throughput screening of regulators. Therefore, in this study, we developed a cellular model and method for high-throughput drug screening targeting TRPV4 channels based on a double mutant(YFP-H148Q/I152L) of the yellow fluorescent protein (YFP) and the calcium-activated chloride channel protein 1, Anoctamin 1 (ANO1). The endogenous expression of TRPV4 in Fischer Rat Thyroid (FRT) cells was determined, TRPV4 ion channel function in FRT cells was verified by electrophysiological techniques, and a TRPV4 cell model co-expressing ANO1 and YFP-H148Q/I152L was constructed. The model was verified to sensitively detect changes in intracellular Ca2+ concentration using membrane clamp experiments and fluorescence quenching kinetics, and the function of the TRPV4 cell model was examined under different temperatures and concentrations of TRPV4 regulators. The model was evaluated to perform well in high-throughput screening.
Publisher
Research Square Platform LLC