Study On The Molecular Mechanism Of The Activation Of TMEM16A Channel By CX And The Physiological Effects Of CX

Ca²⁺-activated chloride channels（CaCCs）were first found in the Xenopus oocyte,where CaCCs are important for blocking polyspermy.It has been identified that TMEM16A is the molecular basis of CaCCs.TMEM16A is activated by intracellular Ca²⁺,and the current is characterized by Ca²⁺and voltage dependent activation,strong outward rectification.TMEM16A is widely distributed in epithelial cells,olfactory sensory neurons,nociceptive neurons,airway smooth muscle cells,vascular smooth muscle cells,and gastrointestinal stromal smooth muscle cells.The dysfunction of TMEM16A is associated with a variety of diseases,including gastrointestinal motility disorders,cystic fibrosis,Sjogren’s syndrome,hypertension,and a variety of cancer.Therefore,TMEM16A has become a specific target for the treatment of these diseases.Screening modulators of TMEM16A and studying its mechanism of physiological and pathological effects on TMEM16A related diseases has become the current research focus.At present,the TMEM16A modulators that have been screened are mostly chemical compounds,which have disadvantages of strong cytotoxicity and low bioavailability.Compared with chemical synthesis products,natural Chinese medicine small molecular compounds have advantages of mild property and low toxicity.In this study,we mainly committed to screening out a modulator from natural Chinese medicines that specifically regulates TMEM16A channel and exploring the regulation of the modulator on diseases caused by TMEM16A dysfunction.In this paper,TMEM16A ion channels were used as the research object.We screened canthaxanthin（CX）for its activated activity on TMEM16A ion channels by fluorescence microscopy imaging combined with patch clamp electrophysiological technique.In addition,we used molecular docking method,site-directed mutagenesis technique combined with patch clamp electrophysiological experiment technique and pharmacological experiments to study on the molecular mechanism of the activation of TMEM16A channel by CX and the physiological effects of CX.The main conclusions were listed in the following three parts:（1）It was found from 13 candidate natural Chinese medicine small molecule compounds that canthaxanthin can activate TMEM16A channel in a concentration-dependent and specific manner with an EC₅₀ value of 5.70±0.72μM.（2）Canthaxanthin is an activator of TMEM16A channel with low cytotoxicity and good biocompatibility.K769 plays an important role in the binding of canthaxanthin to TMEM16A channel,and the TMEM16A channel mutant（K769N）cannot be activated by canthaxanthin.（3）Canthaxanthin can promote the contraction of guinea pig ileum muscle by activating TMEM16A channel,which can be completely inhibited by TMEM16A channel inhibitor(Ca CC_inh-A01).Canthaxanthin is expected to be used in the treatment of gastrointestinal motility.