Structural Mechanism Of Thermal Activation Of Bat TRPV1

Objective:The transient receptor potential vanilloid 1?TRPV1?is a non-selective cation channel which can be activated by heat.As a temperature sensor,TRPV1 plays a crucial role in ambient temperature detection and thermal homeostasis.Although the pore region of TRPV1 has been identified to be involved in the heat-activated gating process,its exact molecular mechanism has not been fully elucidated.We found that a special mammal,fruit bat,which core body temperature range fluctuates more than that of human,this distinctive physiological characteristic may be related to the different responses of TRPV1of the two species to temperature.Therefore,we intend to compare the heat activation characteristics of TRPV1 in bats and humans by electrophysiological recording,and explore the mechanism of the pore region of TRPV1 how to sense high temperature.Methods:In this study,we first transiently transfected TRPV1 channel into HEK293cells with liposome,then carried out the electrophysiological heat activation experiments with human and fruit bat wild-type TRPV1 using inside-out mode to obtain the heat activation temperature threshold and Q₁₀ value.After quantifying the temperature thresholds,which are significantly different,of the two channels,we analyzed the amino acid sequences of the two channels.And two groups of chimeric channels between two kinds of wild-type TRPV1 with interchangeable pore region structure,referring to the comparison results,were prepared for electrophysiological experiments.It was found that the exchanges of pore region were accompanied with temperature threshold change.In order to determine whether the temperature threshold of heat activation are related to the amino acid distribution in the pore region of the two channels,we prepared single-point mutant and three-points mutant on different key amino acid sites according to the alignment results of the pore turret,and measured the thermal activation characteristic parameters of the mutants.Results:1)Patch clamp electrophysiological experiments identified that fbV1 can be activated by capsaicin and heat,while the heat activation temperature threshold of fbV1was lower than that of hV1?42.2±0.2?and 40.2±0.2?,respectively?.There was no significant difference between the Q₁₀ values of the two wild-type TRPV1.2)The chimeric channels of fbV1 with the exchange of pore turret exhibited the increased temperature threshold?42.0±0.3??.Correspondingly,the heat activation threshold of the hV1embedded with the pore turret of fbV1 decreased to 40.1±0.5?.Moreover,capsaicin can still be used as a powerful agonist to cause the strong opening of chimeric channels.3)Through sequence alignments between fbV1 and hV1,we found that there were three pairs of special amino acid residues exchanged in the pore turret region.After the three sites were mutated in turn,the thermal activation thresholds of fbV1 mutants moved in varying degrees.Similarly,the thermal activation thresholds of mutation channels based on hV1changed comparing with that of wild type hV1.And the mutation channels still can be strongly activated by capsaicin.4)Furthermore,the results of triple mutation channels were similar to the chimeric TRPV1 channels.The threshold value of thermal activation of TRPV1 channel in triple mutant hV1 was 40.3±0.2?,similar to that in wild type fbV1,and the threshold value of thermal activation of triple mutant fbV1 was 41.9±0.2?.However,there was no significant difference in Q₁₀ value,and the triple mutant channels were still strongly activated by capsaicin.Conclusion:1)Wild-type hV1 and fbV1 can be thermally activated at different temperatures,but there is no significant difference in temperature sensitivity(Q₁₀)between the two wild-type TRPV1.2)The interchange of pore turret structure made the heat activation temperature threshold exchange between hV1 and fbV1.3)The S609,P614,and S624 residues located in the pore turret region of fbV1 are the key elements participating in the heat activation process of TRPV1.4)The modifications of pore turret structure make no significant effect on capsaicin activation of TRPV1.