The Identification And Function Of Mouse Myocardium TMEM16A

Since 1983 Barish had first indentified the calcium-activated chloride channels(CaCCs)in Xenopus oocytes,CaCCs have attracted much attention.CaCCs are widely distributed in many tissues and involved in numerous physiological processes,such as sensory transduction,regulating the excitability of neuron and myocardium,epithelial secretion and even participating in cell cycle and cell proliferation.However the gene(s)encoding CaCCs is unknown,until 2008,three independent groups identified transmembrane protein 16A(TMEM16A)as a strong candidate gene to encode(or at least a major component of)a calcium-activated chloride channel.TMEM16A is a member of the Anoctamin family of membrane proteins with voltage-and calcium-dependent chloride channel activity,which consists of ten components that share a highly conserved structure with eight transmembrane domains and cytosolic amino-and carboxy-termini domains.Studies have reported that calcium activated directly TMEM16A to generate the chloride currents.Athrough CaCCs has been evident in the heart and play important roles in maintaining cardiac excitability,the molecular basis underlying cardiac CaCCs is not clarified so far.Thus,this study analyzed the biophysical characteristics of CaCCs and the relationship with TMEM16A by using electrophysiological recording combined with cellular and molecular examinations in embryonic hearts ranging from E8.5 to E18.5 as well as postnatal and adult.The results are as follows:The previous studies reported that TMEM16B which was a member of Anoctamin family was a calcium-activated chloride channel in many tissues,but PCR analysis showed that TMEM16A played a primary role in mouse heart.RT-qPCR analysis showed that mRNA of TMEM16A from embryonic mouse heart was detectable at E8.5,at which the heart starts beating regularly,and its level reached peak at E10.5.From E14.5 onward,mRNA of TMEM16A was gradually decreased and maintained at a relative consistent level.Western blot analysis indicated that the expression of TMEM16A gradually increased during embryonic development.Immounostaining assay with heart slices demonstrated that TMEM16A distributed on the plasmalemma and in the cytoplasma.Whole-cell patch clamp recordings revealed that ICl,Ca was recorded in neonatal mouse cardiomyocytes,showing a voltage dependent and niflumic acid(NFA),an anion transport blocker,sensitive properties.The relationship of the ECl values against different external Cl-concentrations is consistent with the channel behavior as a Cl-sensitive channel.It is demonstrated that the currents are ICl,ca which are blocked by NFA(IC50=16.94204?M)and caffeine.What's more,the ICl,Ca is also blocked by T16Ainh-AO1(IC50=2.1497?M),specific inhibitor of TMEM16A,sensitive properties.It shows that the heart weight index and body weight of TMEM16A knockout mouse are below the contrast group.But the heart weight to body weight ratio is just the opposite.Then the experiment found that the expression of ANP and BNP,as marker of myocardial hypertrophy,are up regulation.In summary,TMEM16A was CaCCs to generate the chloride currents.TMEM16A showed a dynamic expression pattern during mouse heart development,indicating that they could play an important role in cardiac excitability.