Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains 英文参考文献.docVIP

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Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains 英文参考文献.doc

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Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains 英文参考文献

Near-MembraneDynamicsandCaptureofTRPM8 ChannelswithinTransientConfinementDomains LuisA.Veliz1,3,CarlosA.Toro1,JuanP.Vivar1,LuisA.Arias1,JeniferVillegas1,MaiteA.Castro2, SebastianBrauchi1* 1InstitutodeFisiologia,FacultaddeMedicina,UniversidadAustraldeChile,CampusIslaTeja,Valdivia,Chile,2InstitutodeBioquimica,FacultaddeCiencias,Universidad AustraldeChile,CampusIslaTeja,Valdivia,Chile,3InstitutodeIngenieriaInformatica,FacultaddeCienciasdelaIngenieria,UniversidadAustraldeChile,CampusIslaTeja, Valdivia,Chile Abstract Background:Thecoldandmentholreceptor,TRPM8,isanon-selectivecationchannelexpressedinasubsetofperipheral neuronsthatisresponsibleforneuronaldetectionofenvironmentalcoldstimuli.Itwaspreviouslyshownthatmembersof the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process.Singleparticletracking(SPT)isausefultoolforprobingtheorganizationanddynamicsofproteinconstituentsin theplasmamembrane. Methodology/PrincipalFindings:WeusedSPTtostudythereceptordynamicsanddescribemembrane/near-membrane behaviorofparticlescontainingTRPM8-EGFPintransfectedHEK-293TandF-11cells.Cellswereimagedusingtotalinternal reflectionfluorescence(TIRF)microscopyandthe2Dand3DtrajectoriesofTRPM8moleculeswerecalculatedbyanalyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel,mostTRPM8particlesmoveinnetworkcoveringthePM,periodicallylingeringfor2–8sinconfinedmicrodomains ofabout800nmradius.Removingcholesterolwithmethyl-beta-cyclodextrin(MbCD)stabilizesTRPM8motioninthePM andiscorrelatedwithlargerTRPM8currentamplitudethat