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dc.contributor.authorGarcia, Alvaro
dc.contributor.authorLev, Bogdan
dc.contributor.authorHossain, Khondker R.
dc.contributor.authorGorman, Amy
dc.contributor.authorDiaz, Dil
dc.contributor.authorPham, T. H. Nguyen
dc.contributor.authorCornelius, Flemming
dc.contributor.authorAllen, Toby W.
dc.contributor.authorClarke, Ronald J.
dc.date.accessioned2019-09-10T01:44:16Z
dc.date.available2019-09-10T01:44:16Z
dc.date.issued2019-02-15
dc.identifier.citationGarcia, A., Lev, B., Hossain, K. R., Gorman, A., Diaz, D., Pham, T. H. N., … Clarke, R. J. (2019). Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment. Journal of Biological Chemistry, 294(15), 5956–5969. https://doi.org/10.1074/jbc.ra118.006223en_AU
dc.identifier.urihttp://hdl.handle.net/2123/21055
dc.description.abstractCholesterol’s effects on Na+,K+-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na+,K+-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol’s effect on Na+,K+-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-β-cyclodextrin (mβCD). The mβCD-treated Na+,K+-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa+3 → E2P, i.e. phosphorylation by ATP and Na+ occlusion from the cytoplasm) and K+ deocclusion with subsequent intracellular Na+ binding (E2K+2 → E1Na+3). However, cholesterol depletion did not affect the observed rate constant for K+ occlusion by phosphorylated Na+,K+-ATPase on the extracellular face and subsequent dephosphorylation (E2P → E2K+2). Thus, partial reactions involving cation binding and release at the protein’s intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na+,K+-ATPase into the E2 state, requiring a slow E2 → E1 transition before the phosphorylation. Molecular dynamics simulations of Na+,K+-ATPase in membranes with 40 mol% cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further disclosed state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na+,K+-ATPase steady-state activity.en_AU
dc.description.sponsorshipAustralian Research Council, National Health and Medical Research Council (Australia)en_AU
dc.language.isoen_AUen_AU
dc.publisherAmerican Society for Biochemistry and Molecular Biologyen_AU
dc.relationARC DP121003548, ARC DP150101112, ARC DP170101732, NHMRC APP1104259en_AU
dc.rightsThis research was originally published in the Journal of Biological Chemistry. Alvaro Garcia, Bogdan Lev, Khondker R. Hossain, Amy Gorman, Dil Diaz, Thi Hanh Nguyen Pham, Flemming Cornelius, Toby W. Allen and Ronald J. Clarke. Cholesterol depletion inhibots Na+,K+-ATPase activity in a near-native membrane environment. J. Biol. Chem. 2019; 294:5956-5969. © the American Society for Biochemistry and Molecular Biologyen_AU
dc.subjectlipid-protein interactionsen_AU
dc.subjectsteady-state activityen_AU
dc.subjectpartial reaction kineticsen_AU
dc.subjectmethyl-beta-cyclodextrinen_AU
dc.subjecteosinen_AU
dc.subjectmolecular dynamicsen_AU
dc.subjectcation pumpen_AU
dc.subjection transferen_AU
dc.subjectelectrochemical potentialen_AU
dc.subjectreconstituted membraneen_AU
dc.titleCholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environmenten_AU
dc.typeArticleen_AU
dc.subject.asrcFoR::030403 - Characterisation of Biological Macromoleculesen_AU
dc.identifier.doiDOI 10.1974/jbc.RA118.006223
dc.type.pubtypePost-printen_AU


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