The Mechanism Of TRPM2 Channel Gating By Calcium

Transient receptor potential melastatin 2(TRPM2)channel is a Ca2+-permeable,non-selective cation channel,which is widely expressed in various cells,such as neurons,microglia,pancreatic ?-cells,monocytes,macrophages and endothelial cells.Recent studies show that TRPM2 channel plays a role in many physiological and pathological processes,including warmth sensing,inflammation,ischemia injury,neurodegenerative diseases,diabetes and tumors.The major endogenous ligand of TRPM2 channel is adenosine diphosphate ribose(ADPR),which is the metabolite of nicotinamide adenine dinucleotide(NAD+).TRPM2 channel can also be activated by H2O2,so it is regarded as an important oxidative stress sensor in our body.Moreover,as an important signaling molecule,Ca2+not only can activate TRPM2 channel in synergy with ADPR,but also can open the channel by itself.However,the mechanism of TRPM2 channel gating by Ca2+ is still unclear.To explore whether there is a Ca2+ binding site in TRPM2 channel,we predicted two potential EF-loops(D267-D278,D288-E298)in the N-terminus of human TRPM2 channel(hTRPM2),using bioinformatics methods.Through alanine mutagenesis screen,we made functional electrophysiological assay of these two motifs,and found that alanine mutations at residues of high conservation dramatically affected channel currents(For example,D267A,D278A,D288A and E298A).Further functional assay of multiple substitutions at these highly conserved residues indicated that,as for D267-D278,the negative charge of D267 and the side chain of D278 were critical for Ca2+-induced hTRPM2 channel activation.Moreover,G272R and G272I mutants significantly reduced hTRPM2 channel currents,suggesting that the increasement of the side chain length of G272,affected hTRPM2 channel activation by Ca2+.Most of the mutants at residues in D288-E298 led to the complete loss of channel currents,without the characteristic that only the conserved sites affected channel function.Meanwhile,biotinylation assay illuminated that these mutants had no effect on membrane trafficking of the channel.Further single-channel recordings suggested that the changes of channel currents by these mutants were not result from their influence on single channel conductance.Our data suggest that D267-D278 serves as an EF-loop which participates in TRPM2 channel activation by Ca2+,while D288-E298 doesn't mediate Ca2+bindingMoreover,through sequence alignment of the transmembrane segments of TRPM2 channels from different species,and the further alignment among TRPM2,TRPM4,TRPM5 channels,we found a potential Ca2+binding site in the S2,S3 transmembrane segments domain(S2-S3 domain)of them.More importantly,a recent study resolved the structure of Nematostella vectensis TRPM2 channel(nvTRPM2),and discovered a Ca2+binding site,which is constructed of four residues:glutamate,glutamine,asparagine,aspartate,in its S2-S3 domain.Nonetheless,the effects of these residues on TRPM2 channel gating is unknown.Through mutation techniques and electrophysiological recordings,we systematically tested the influence of single site mutation on the activation and inactivation processes of nvTRPM2 channel and hTRPM2 channel.Our data display that:1)E893A nvTRPM2 can hardly by activated,while E843A hTRPM2 behaves the same as wild type;2)Except the positively-charged lysine substitution at glutamine,other mutants at this site show milder effects on channel activation,while they accelerate channel inactivation;3)Both of alanine and lysine mutations at asparagine make the channel hardly be activated.Although the aspartate mutation here has no influence on channel activation,it increases the inactivation rate of the channel to a large extent;4)all kinds of mutants at aspartate have significant effects on the activation of nvTRPM2 channel and hTRPM2 channel.All above results indicate that the negatively-charged residues in these four sites(glutamate and aspartate)mainly affect the channel activation,rather than inactivation.The charge-neutralizing residues(glutamine and asparagine)exhibit milder effects on channel activation while they facilitate channel inactivation.In summary,our research not only identify a novel EF-loop which is critical for Ca2+-induced TRPM2 channel activation,in the N-terminus of the channel,but also discover the influence on channel activation and inactivation which is induced by Ca2+binding with the S2-S3 domain of TRPM2 channel,proposing new mechanisms about TRPM2 channel gating by Ca2+.Together with the discoveries of other research groups,our results fully manifest that the gating progress of TRPM2 channel by Ca2+is very precise and complex,and we also provide new thinking and experimental evidence for understanding the molecular mechanisms of physiological and pathological processes mediated by TRPM2 channel.