Upregulation Of TRPV2 Channel Functions Via Mg²⁺-mediated Dephosphorylation

Transient receptor potential?TRP?channels represent a large and diverse family of non-selective cation channels,and they are involved in mediating sensory transduction of pain,cold,heat,mechanical,osmotic or noxious stimuli.In mammals,the TRP superfamily is subdivided into six families named TRPC,TRPV,TRPA,TRPM,TRPP and TRPML.TRPV2 belongs to thermo-sensitive TRP channels and can be activated by heat??52??,and is a nociceptive receptor.Although TRPV2 channel is widely expressed and functions in tissues and organs,the function mechanisms still remain unclear,this is because that the heat activation threshold of TRPV2 channel can be hardly achieved under physiological conditions,and specific activators and inhibitors of TRPV2 channel have not yet been found,which bring difficulties to researches on TRPV2 channel.TRPV2 is non-selective cation channel,opened TRPV2 channels can permeate divalent cations(Ca²⁺,Mg²⁺)and monovalent cation?Na⁺,K⁺?,divalent cations flux into cells through TRPV2 channels and regulate TRPV2 channel' activity via mediating signal pathways,which play an important role in feedback regulation of ion channels' functions.For example,Ca²⁺ activate phospholipase C?PLC?signal pathway after fluxing into cells via TRPV2 channel,which lower the level of PIP2 on the cellular membrane and induce TRPV2 channel's desensitivity,and impair the function of TRPV2 channel.Our pre-experiment results showed that physiological concentration Mg²⁺ effectively potentiated the currents of TRPV2 channel,which was different with downregulation of Ca²⁺ on TRPV2 channel.In this study,we explored function mechanisms of Mg²⁺ on TRPV2 channel,and acquired main results were as follows:1)Functions of TRPV2 channel could be potentiated by extracellular Mg²⁺ at a dose-dependent manner.We used patch clamp and calcium imaging to observe the modulation of Mg²⁺ on 2-APB induced TRPV2 currents,and found Mg²⁺potentiated the currents of TRPV2 channel and this potentiation was at a dose-depended manner.What's more,We also observed that Mg²⁺ can enhance heat-induced TRPV2 currents,which means the potentiation of Mg²⁺ on TRPV2 channel is not 2-APB-depended.2)Mg²⁺ enhance the currents of TRPV2 channel from the intracellular side.In the patch-clamp experiment,we used low concentration of 2-APB to activate TRPV2 channels which reduced opened channels and made few Mg²⁺ enter into cells,then we observed that the potentiation of Mg²⁺ on TRPV2 channel was disappeared.What's more,chelating agents of Mg²⁺,EDTA and EGTA,could effectively block the potentiation when applied from intracellular side.Thus,the results showed that the potentiation of Mg²⁺ on TRPV2 channel was from intracellular side.3)S526 and T522 in S5-S6 linker played an important role in the regulation of Mg²⁺on TRPV2 channel.Since the two sites are putative phosphorylation sites,we explored the regulations of phosphorylation and dephosphorylation on TRPV2 channel.4)Phosphorylation could modify TRPV2 channel and downregulate TRPV2 channel's activity,but Mg²⁺-mediate dephosphorylation could upregulate TRPV2 channel's activity.We used PKC activator,PMA,to stimulate TRPV2 channel,and observed that PMA reduced the sensitivity of TRPV2 channel to 2-APB;on the contrary,the potentiation of Mg²⁺ on TRPV2 was disappeared when we used phosphorylation inhibitors to treat cells.Thus,we confirmed that Mg²⁺ could potentiate the currents of TRPV2 channel through promoting phosphatase to catalyze TRPV2 channel to be dephosphorylated.5)Mg²⁺ metabolic balance and nociceptive sensation which was mediated via TRPV2 channel.Based on the above results,since TRPM7 channel was responsible for permeating Mg²⁺,we coexpressed TRPM7 channel and TRPV2 channel in cells,the improvement of intracellular Mg²⁺ through TRPM7 channel could potentiate the function of TRPV2 channel.In conclusion,our results showed that phosphorylation and dephosphorylation played an important role in the regulation of TRPV2 channel's activity,which provided effective methods for modulating TRPV2 channel,and revealed Mg²⁺play an vital role in the nociceptive sensation through regulating TRPV2 channel activity.