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Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment.

ABSTRACT: Cholesterol'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-?-cyclodextrin (m?CD). The m?CD-treated Na+,K+-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa3 + ? E2P, i.e. phosphorylation by ATP and Na+ occlusion from the cytoplasm) and K+ deocclusion with subsequent intracellular Na+ binding (E2K2 + ? E1Na3 +). 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 ? E2K2 +). 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 indicated 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.

SUBMITTER: Garcia A

PROVIDER: S-EPMC6463725 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment.

Garcia Alvaro A Lev Bogdan B Hossain Khondker R KR Gorman Amy A Diaz Dil D Pham Thi Hanh Nguyen THN Cornelius Flemming F Allen Toby W TW Clarke Ronald J RJ

The Journal of biological chemistry 20190215 15

Cholesterol'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 deplet ...[more]

PMID: 30770471

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Project description:A single Na(+)/K(+)-ATPase pumps three Na(+) outwards and two K(+) inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na(+) than K(+) generates outward current across the cell membrane. Less well understood is the ability of Na(+)/K(+) pumps to generate an inward current of protons. Originally noted in pumps deprived of external K(+) and Na(+) ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K(+) and Na(+) concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na(+) release from phosphorylated Na(+)/K(+) pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na(+)/K(+) pumps that enables proton import is not required for completion of the 3 Na(+)/2 K(+) transport cycle. However, the back-step occurs readily during Na(+)/K(+) transport when external K(+) ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na(+)-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na(+) and K(+) ions that passes through binding site II. The inferred occurrence of Na(+)/K(+) exchange and H(+) import during the same conformational cycle of a single molecule identifies the Na(+)/K(+) pump as a hybrid transporter. Whether Na(+)/K(+) pump-mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

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Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment.

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Cholesterol depletion inhibits Na<sup>+</sup>,K<sup>+</sup>-ATPase activity in a near-native membrane environment.

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