Functional Regulation Of TRPV1 Channel Through Ca²⁺-dependent Tachyphylaxis And SUMOylation Modification

The Transient Receptor Potential Channel subfamily V member 1(TRPV1)channel belongs to the TRP channel family,and is widely expressed in various tissues of the body and can respond to a variety of stimuli.The TRPV1 channel is involved in important physiological activities such as pain perception,body temperature maintenance and osmotic pressure regulation.Its abnormal function may cause hyperthermia,osmotic pressure abnormality and some inflammatory or neurological hyperalgesia.Therefore,it is of great physiological and pathological significance for in-depth study of the structure and function of the channel.Among all of the studies,the role of TRPV1 channel in the nociception is our focus.In the body,hyperalgesia or neuropathic pain caused by development of diseases such as tissue damage,inflammation and tumors is often accompanied by up-regulation of TRPV1 function.Calcium-dependent desensitization of the TRPV1 channel is one of its important features.The neurosensory organs entering the desensitized state are no longer sensitive to other noxious stimuli including capsaicin,or entering the pain refractory period.Specific induction of nociceptive nerve into the pain refractory period is one of the important means of clinical analgesia.The desensitization of TRPV1 channel is divided into rapid desensitization and tachyphylaxis.It is reported that the rapid desensitization of TRPV1 channel is related to the phosphorylation modification,the interaction with calmodulin and the change of PI(4,5)P₂ level on the membrane,but the mechanism underlying tachyphylaxis is still unclear.Our preliminary experimental results have shown that the tachyphylaxis of TRPV1 channel is strength-memorizing,that is,the degree of desensitization is related to the stimulation intensity.The first part of this thesis is aimed at the mechanism underlying tachyphylaxis of TRPV1 channels.The following results were obtained:1)Through the use of exogenous expression and endogenous Dorsal Root Ganglia(DRG)in combination with whole-cell patch clamp recording,it was found that the tachyphylaxis of TRPV1 channel is stimulation intensity-dependent,and the higher the stimulation intensity appled,the more severe the degree of desensitization.2)By successful construction of TRPV1-p Hluorin plasmid which resembled wild type TRPV1 channel in the function,and by using light sheet imaging,the endocytosis-exocytosis trafficking process of TRPV1 protein under different stimulation intensity was observed;3)Throuh the application of dual channel fluorescence imaging to simultaneously observe the strength-memorizin tachyphylaxis of TRPV1 and dynamics of the intracellular Ca²⁺concentratios,it was showed that the intracellular Ca²⁺concenrations increased more and the high level of intracellular Ca²⁺concenrations persisted longer under higher stength stimulation,resulting in more severe desensitization of TRPV1,suggesting that the intracellular calcium signal intensity determined the amount of intracellular trafficking of TRPV1 and also the time required to recirculate back onto the cell membrane;4)By whole-cell patch clamp electrophysiological recording it was shown that when ATP was added into the pipette solution,the current response of TRPV1 channel can be recovered by pausing for a period of time after desensitizaiton.The greater the stimulation intensity used for desensitization,the longer the recovery time is required.5)Finally through the screening of the transport-related protein Synaptotagmin(Syt)by protein interaction and by functional screening,it was found that Syt1 and Syt7 play an important role in the Ca²⁺regulated cyclic trafficking of TRPV1.Syt1 located near the cell membrane specifically regulates the rapid transport process of the TRPV1 channel,while Syt7located in the cytoplasm regulates the slow transport process of TRPV1 channel under high-intensity stimulation.Our results well reveal the process of strength memorizing tachyphylaxis of the TRPV1 channel.Small ubiquitin-like modification(SUMOylation)is a means of post-translational modification of proteins during which the SUMO molecule covalently binds to the lysine residue of target protein.SUMOylation is involved in the regulation of vesicle transport,synaptic function and pain sensation in sensing neurons,and thus plays an important physiological function in the body.Recent studies have shown that SUMOylation can regulate the function of some nociceptive ion channels.Our preliminary studies have shown that peripheral inflammation induced by injection of Carageenan or Complete Freund's Adjuvant resulted in increasing thermal hyperalgesia behavior of wild type mice,while leaving no significant influence on Trpv1^-/-mice.And the immunoblotting results showed the up-regulation of TRPV1 SUMOyaltion levels in the DRG of mice under inflammatory state.To explore the effect of SUMOylation on TRPV1 channel-mediated nociceptive thermal response,we designed a series of experiments that yielded the following results:(1)SUMOylation significantly and selectively reduced the temperature activation threshold of TRPV1 channel while not affecting the sensitivity of TRPV1 channel to acidic p H,capsaicin and voltage stimulation;(2)SUMOylation modification of rat-and mouse-derived TRPV1 channel can affect the thermal threshold of both channels in a simiar way.(3)In the combination of bioinformatics and biochemical experiments,it was found that the lysine residue at position 822 of TRPV1 channel is the main site of SUMOylation modification,which was also comfirmed by our whole-cell patch clamp recordings.Our results explained the cause of hyperthermia in the body induced by peripheral inflammation and elucidate the mechanism by which SUMOylation up-regulates the thermal sensitivity of the TRPV1 channel.In summary,this paper clarifies the mechanism underlying Ca²⁺-dependent strength-memorizing tachyphylaxis of TRPV1 channel,and reveals the regulation mechanism of SUMOylation on thermal hyperalgesia mediated by TRPV1 channels,enriching the understanding of TRPV1 structure and function.The relavant research results provide a new intervention strategy for pain treatment targeting TRPV1 channels.