Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening 英文参考文献.docVIP

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Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening 英文参考文献.doc

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Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening 英文参考文献

Fluorescence-TrackingofActivationGatinginHuman ERGChannelsRevealsRapidS4MovementandSlow PoreOpening ZeinebEs-Salah-Lamoureux1.,RobertFougere1.,PingYuXiong1,GailA.Robertson2,DavidFedida1* 1Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada, 2Department of Physiology, UniversityofWisconsin-MadisonSchoolofMedicine,Madison,Wisconsin,UnitedStatesofAmerica Abstract Background:hERGchannelsarephysiologicallyimportantionchannelswhichmediatecardiacrepolarizationasaresultof theirunusualgatingproperties.Theseareveryslowactivationcomparedwithothermammalianvoltage-gatedpotassium channels,andextremelyrapidinactivation.Themechanismofslowactivationisnotwellunderstoodandisinvestigated hereusingfluorescenceasadirectmeasureofS4movementandporeopening. Methods and Findings: Tetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltagesensormovement,andchannelopeningandclosinginhERGchannels.Endogenouscysteines(C445andC449)in the S1–S2 linker bound TMRM, which caused a 10mV hyperpolarization of the VK of activation to 227.562.0mV, and showedvoltage-dependentfluorescencesignals.SubstitutionofS1–S2linkercysteineswithvalinesallowedunobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with V =237.861.7 mV, and K,1 VK,2=43.567.9mV.Thefirstphase,VK,1,was,20mVnegativetotheconductance-voltagerelationshipmeasuredfrom ionic tail currents (G-VK=218.361.2mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (VK=234.461.5mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (VK=220.661.2, k=11.4mV) and L520C quenching during depolarization (VK=226.861.0, k=13.3mV) matched t