The Research About The Competed Binding Sites In TMEM16A Calcium Activated Chloride Channel For PIP₂ And Honokiol

Objective: Calcium-activated chloride channels（Ca CCs）are a kind of very important membrane proteins that regulate cellular functions.They are widely distributed in vascular endothelium,respiratory tract endothelium,pancreatic acinus,breast cancer and other tissues and organs,they regulate a variety of physiological functions and pathological processes in the body.TMEM16 A as a voltage dependence outwardrectifying characteristic of calcium activated chloride channel,who plays an important role in a variety of pathologic physiology in the process,such as to avoid multiple fertilized oocytes,mediated the liquid secretion of epithelial cells,mediated smooth muscle contraction,mediated sensory signal transduction,and promote the occurrence of tumor and cell migration,etc.With the gradual deepening of research on the function of TMEM16 A,the current focus is mainly on the use of TMEM16 A as a drug target and the functional regulation mechanism of TMEM16 A.This study usedin phosphatidyl inositol 4,5-diphosphate（PIP₂）and Honokiol as activator and inhibitor compete TMEM16 A channel respectively,application of amino acid fixed point mutation and electrophysiological technique of phosphatidyl inositol 4,5-diphosphate（PIP₂）and Honokiol with TMEM16 A channel binding sites for research,and provide a structure to develop TMEM16 A specificity drugs.Honokiol is an active ingredient of Magnolia officinalis,which has obvious and lasting central muscle relaxation,central nervous inhibition,anti-inflammatory,anti-bacterial,anti-pathogenic microorganism,anti-ulcer,anti-oxidation,anti-aging,anti-tumor,cholesterol-lowering and other pharmacological effects.Our research group’s previous research found that the Honokiol has inhibitory effect on TMEM16 A current in whole-cell recording mode,application of amino acid,double locus mutation point unit of Honokiol binding site study,confirmed that TMEM16 A R429and K430 may be the key in the sequence of binding sites,and H Criss.Hartzell reports PIP₂ and TMEM16 A of binding sites such as the same,but whether the two can competitively bind to the same site is unclear.In this study,under the inside-out recording mode,we will give Honokiol and PIP₂ separately and sequentially to study whether the two drugs competitively bind to the same site.Phosphatidylinositol 4,5-diphosphate（PIP₂）is a kind of phospholipid,which accounts for less than 1% of membrane phospholipids,but has very complex functions.This low abundance polyphosphoinositol lipid can be hydrolyzed by phospholipase C（PLC）activated by GQ type G protein-coupled receptor to inositol triphosphate（IP3）and diglyceride（DG）to participate in signal transduction.IP3 binds and excites the IP3 receptor on the endoplasmic omentum to trigger the release of Ca2+,and the released Ca2+ binds to the TMEM16 A protein causing the opening of the conformational change-mediated channel.PIP₂ is a negatively charged polar molecule,and its target protein binding site is usually a positively charged amino acid.A well-known PIP₂ binding site is the Pleckstrin Homology（PH）domain,which binds PIP₂ to a pocket of basic amino acids that are not adjacent in the first order sequence,but are very close together in the folded protein.Studies have shown that there are multiple PIP₂ binding sites in TMEM16 A structure,which provides a basis for the study of the functional regulation mechanism of TMEM16 A according to the different binding sites.In this study,the inside-out recording method was mainly used by patch clamp,PIP₂ and Honokiol were administered separately and sequentially to wild-type TMEM16 A channel and mutant TMEM16 A channel（R429 and K430）,The aim is to study the key binding sites of the functional regulation of TMEM16 A by both of them and whether there are competitive binding sites of the same sites,so as to provide a solid theoretical basis for targeting the design and optimization of drugs acting on TMEM16 A channels.Methods: In this study,plasmid transformation and extraction technology were used to amplify and extract m TMEM16a-EGFP plasmid,which was mutated into R429 A and K430 L unit point mutant plasmid and R429A-K430 L double site mutantplasmid by PCR experiment technology.Rely on transient transfection technique,respectively in HEK293 cells transfection m TMEM16A-EGFP plasmid,R429 A mutation plasmid,K430L mutation plasmid,R429A-K430 L double locus mutation plasmid,the patch clamp Inside-out records,for the membrane,the 270 n M calcium concentration under the experimental conditions,record PIP₂ and Honokiol dosing and delivery order separately for transfection of wild-type and mutant TMEM16 A current and the influence of the changes.Experimental results: Using the Inside Out patch-clamp recording method,it was found that PIP₂ had different activation effects on the TMEM16 A current of HEK293 cells transfected with m TMEM16A-EGFP plasmid,R429 A and K430 L unit point mutant plasmid and four plasmids of R429A-K430 L double site mutant plasmid under the condition of calcium concentration at 270 n M.The activation effect of wild-type was higher than that of the two single mutants,which were 78.6%（WT）,19.8%（R429A）,12.8%（K430L）and 8.16%（R429A-K430L）,respectively.Similarly,under the same experimental conditions,Honokiol showed the inhibition effect on TMEM16 A current,and the inhibition rate of wild type was higher than that of single mutant and double mutant,and the inhibition rates were 61.6%（WT）,46.6%（R429A）,43.9%（K430L）and 27.1%（R429A-K430L）in order.When PIP₂ and Honokiol were sequentially administered to wild-type HEK293 cells,the inhibitory effect of Honokiol and PIP₂ on TMEM16 A was much lower than that of Honokiol and Honokiol alone.In HEK293 cells transfected with double mutant plasmid,the inhibitory effect of PIP₂ and Honokiol on TMEM16 A current was almost the same as that of Honokiol alone.Conclusion: The study confirmed the activation of exogenous PIP₂ on TMEM16 A and the inhibition of Honokiol on TMEM16 A.After unit point mutation,both activation and inhibition were significantly reduced,which proved that R429 and K430 sites were two important sites among many binding sites of PIP₂,Honokiol and TMEM16 A.And in our laboratory prove that Honokiol and TMEM16 A binding sites including R429 and K430 under the condition of the two,in the wild type of cell found in order to give the two drugs,inhibition rate is far lower than the wild type of Honokiol alone under the condition of Honokiol inhibition rate,at the same time after the double mutation plasmid transfection,inhibition rate almost unchanged after order to give the two drugs,that PIP₂ and Honokiol in R429 and K430 two binding sites competitive relationship.This study provides new viewpoints and research results for the functional regulation of exogenous PIP₂ on the TMEM16 A channel and the mechanism of its combination with Honokiol,and provides new directions and methods for further studies on the specific action sites of other drugs on TMEM16 A.