The Study Of Electro Physiological Properties Based On CaCCs/TMEM16B As Targets For Drug

Calcium-activated chloride channels (CaCCs) are widely expressed in various celltypes and tissues, and involved in important physiological processes, including regulationof cardiac excitability, secretion by exocrine glands, sensory transduction. TMEM16A andTMEM16B have been shown to function as endogenous CaCCs. It has been reported thatTMEM16B composes part of the molecular basis of CaCCs. Due to the vital physiologicaland pharmacological roles of TMEM16B, the electrophysiological characteristics areneeded to resolve.CaCCs dysfunction may lead to certain diseases, such as cystic fibrosis,gastrointestinal disorders, asthma and diarrhea. Screening of TMEM16B-targeted drugs isone of the hot issues in the field. The previous results of our lab have confirmed thatchitosan oligosaccharide(COS) can activate the TMEM16A channels, but their molecularinteractions between TMEM16A and COS have not been understood yet.In this paper, TMEM16B ion channels were studied by patch clamp experiments andconfocal microscopy imaging techniques. Some inspiring results were obtained, listed inthe following three parts:(Ⅰ) Electrophysiological characteristics of TMEM16B channelTMEM16B showed highly similar electrophysiological characteristics to naturalCaCCs, wherein(ⅰ) TMEM16B channel has voltage-dependent and calcium-dependent properties,and the half maximal effective concentration(EC50) of Ca2+to TMEM16B channel is3.3μM.(ⅱ) TMEM16B channel varies greatly for different anions in the permeation rate, andthe permeability order goes as follows: I-> NO-3> Br-> Cl-> gluconate.(ⅲ) ATP can not activate TMEM16B, so it can not be used as a positive control inscreening TMEM16B regulator.(Ⅱ) The activation role of COS to TMEM16B and the molecular mechanismOur previous research demonstrated that COS can be used as an activator ofTMEM16A channel. This thesis shows that COS can also activate TMEM16B channels. Inaddition, the activation mechanism is also discussed here. (ⅰ) COS can activate TMEM16B ion channel activation in a dose dependent manner,with an EC50of28.07μg/ml.(ⅱ) COS activate TMEM16B channels not indirectly, via increasing intracellular Ca2+concentration and the downstream signal transduction pathway, but directly activatedTMEM16B channels.(Ⅲ) Microscopic observation COS transmembrane processIn order to explore the activation mechanisms of COS to TMEM16B channel in detail,we studied the fluorescence-labeled COS transmembrane process with the laser scanningconfocal microscopy.(ⅰ) COS transports into the cell in a temperature-dependent manner. Intracellularfluorescence intensity increases with increasing temperature. It can be deduced that COSendocytosis process requires energy intake.(ⅱ) Increase fluidity of membrane facilitates COS entering the cell. It was found thatmobile calcium carrier A23187can promote COS to transport into the cell. According tothe experimental results, it can be proposed that A23187facilitated phosphatidylserineeversion on the membrane, and then the membrane fluidity increases, thereby promotingCOS endocytosis process.