TMEM16A Participates In Airway Smooth Muscle Contractility And Airway Hyperresponsiveness

Asthma is a common disease,which has a high incidence in children.According to the data from the World Health Organization(WHO),about 235 million people suffer from asthma,and the incidence is increasing.Asthma always associates with chronic airway inflammation,and recurrent attacks of respiratory symptoms such as breathlessness and wheezing.Asthma not only seriously affect the life quality of patients,severe attacks could even cause death.The large patient population makes asthma a global public health burden.So,studying the pathogenesis and searching new methods to control asthma have great social significanceAsthma attacks is caused by the contraction of smooth muscle,which narrows airway,causes respiratory resistance elevation,and results in the breathlessness.Thus,smooth muscle is the end-effector tissue to cause respiratory symptoms.In physiological condition,airway smooth muscle is innervated by vagal nerve fibers,which release acetylcholine(ACh)to contract smooth muscle by acting M type ACh receptors in smooth muscle cells,hence regulate constriction of airway;while in asthmatic condition,the airway smooth muscle is infiltrated with inflammatory cells such as mast cells,hyper-responds to nerve impulse and other stimuli,shows airway hyperresponsiveness(AHR).Although,it is already know that the formation of AHR is highly related to allergic inflammation,the mechanisms underlying are still not clear.The inflammatory cells associated with airway allergic inflammation could secrete large amounts of inflammatory mediators and cytokines.The major function of these mediators is mediating immune reactions.Interestingly,some inflammatory mediators,such as histamine,5-HT,prostanoids,endothlins and cysteiny leukotrienes(CysLTs)could directly contract smooth muscle(We will call them inflammatory constrictors in later paragraphs).But the regulating mechanisms and pathological significance of this kind of contraction is not clear.According to existing literature reports and our previous results,we speculate there may exist some unique regulatory mechanisms in these inflammatory mediators mediated smooth muscle contraction,and these mechanisms may participate in the formation of AHR.The known mechanisms of smooth muscle contraction were descripted as two pathways.In one of them,cell membrane depolarization opens voltage-dependent Ca2+channels(VDCCs),causes Ca2+influx and activates Ca2+-dependent myosin light chain kinase(MLCK)to initiate contraction,this pathway is called as electromechanical coupling;in the other one,agonists activate corresponding G protein coupled receptors(GPCRs),through complex multiple downstream signals(including Ca2+-MLCK axis and Ca2+sensitivity mechanisms)to mediate contraction,this pathway is called as pharmacomechanical coupling.Based on existing theory,GPCRs mediate contraction mainly through inducing sarcoplasmic reticulum(SR)Ca2+release to elevate cytosolic Ca2+concentration([Ca2+]i).However,as early as 1990s,the phenomenon that GPCR agonists could induce depolarization in airway smooth muscle cells had been reported.But,the significance and concrete mechanisms underlying are not clear.We found that ACh and many inflammatory constrictors could induce smooth muscle cell membrane depolarization.So,we hypothesize this kind of depolarization may be one basis of AHR formation.Based on literature reports that Ca2+-activated Cl-(ClCa)channel mediates depolarization inward currents in smooth muscle cells and TMEM16A forms this channel,we further hypothesize that the molecular mechanisms of agonist induced depolarization rely on ClCa channel TMEM16A.To confirm this hypothesis,we respectively measured various inflammatory constrictors induced smooth muscle cell depolarization currents and bronchial smooth muscle contraction force.We also generated TMEM16A conditional knockout mice,systematically studied the signal pathway and contractile characteristics of inflammatory constrictors mediated airway smooth muscle contraction,as well as the role of this pathway in AHR.Our results showed that inflammatory mediators such as 5-HT,U46619 and ET-1 could induce airway smooth muscle depolarization currents and trigger bronchial smooth muscle contraction.When knockout TMEM16A or blocking it by specific inhibitor T16Ainh-A01,the currents eliminated and the contraction was suppressed.We also found 5-HT induced[Ca2+]i elevation was suppressed after deleting TMEM16A.In addition,L-type Ca2+channel inhibitor nifidepine showed a similar inhibitory effect on bronchial smooth muscle contraction,but which didn’t show superimposed effect with TMEM16A knockout.These phenomena indicate that inflammatory constrictors induce smooth muscle depolarization through activating TMEM16A,hence activate VDCCs,promote Ca2+influx and mediate airway smooth muscle contraction.We think this mechanism is one of the main mechanisms to mediate inflammatory constrictors induced contraction,but just an adjoint mechanism in ACh induced contraction.But even just adjoint mechanism,this mechanism may correlate to asthma attacks triggered by weak nerve activity.Through ex vivo bronchial isometric tension measurement,we observed obvious synergistic effect of inflammatory constrictors with low dose MCh.This synergistic augmentation effect partially relied on the function of TMEM16A.By studying mouse asthma model,we found knockout TMEM16A could significantly inhibit airway resistance elevation.Based on these results,we suggest the regulatory pathway of TMEM16A is one of the basis of asthmatic AHR formation,inflammatory mediators could induce asthma attacks through this pathway.In summary,our research shows that TMEM16A is the ClCa channel in airway smooth muscle cells,which mediates GPCR agonists induced depolarization currents,and links pharmacomechanical coupling to electromechanical coupling by activating VDCCs,and participates in inflammatory constrictors and low concentration M type ACh receptor agonist induced bronchoconstriction.TMEM16A may contribute to AHR,especially the hypersensitivity to stimuli,participate in respiratory resistance elevation in asthma.Our results also suggest TMEM16A as a potential drug target to control asthma symptoms.