Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block 英文参考文献.docVIP

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Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block 英文参考文献.doc

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Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block 英文参考文献

SelectionofInhibitor-ResistantViralPotassiumChannels IdentifiesaSelectivityFilterSitethatAffectsBariumand AmantadineBlock FranckC.Chatelain1.,SabrinaGazzarrini2.,YuichiroFujiwara1,CristinaArrigoni2,CourtneyDomigan1, GiuseppinaFerrara2,CarlosPantoja4,GerhardThiel6,AnnaMoroni2,DanielL.Minor,Jr.1,3,4,5,7* 1Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America, 2Dipartimento di Biologia e Istituto di BiofisicadelConsiglioNazionaledelleRicerche,Universita` degliStudidiMilano,Milan,Italy,3DepartmentofBiochemistryandBiophysics,UniversityofCaliforniaSan Francisco,SanFrancisco,California,UnitedStatesofAmerica,4DepartmentofCellularandMolecularPharmacology,UniversityofCaliforniaSanFrancisco,SanFrancisco, California, United States of America, 5Department of California Institute for Quantitative Biomedical Research, University of California San Francisco, San Francisco, California,UnitedStatesofAmerica, 6TechnischeUniversita¨t Darmstadt,Institutefu¨rBotanik,Darmstadt,Germany, 7PhysicalBiosciencesDivision,LawrenceBerkeley NationalLaboratory,Berkeley,California,UnitedStatesofAmerica Abstract Background:Understandingtheinteractionsbetweenionchannelsandblockersremainsanimportantgoalthathasimplications fordelineatingthebasicmechanismsofionchannelfunctionandforthediscoveryanddevelopmentofionchanneldirecteddrugs. Methodology/PrincipalFindings:Weusedgeneticselectionmethodstoprobetheinteractionoftwoionchannelblockers, bariumandamantadine,withtheminiatureviralpotassiumchannelKcv.SelectionforKcvmutantsthatwereresistantto eitherblockeridentifiedamutantbearingmultiplechangesthatwasresistanttoboth.ImplementationofaPCRshuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiologicalandbiochemicalassaysrevealedadist