Odorous chemical perturbations of (Na+ + K+)-dependent ATPase activities. Effects on native and lipid-substituted preparations from individual turbinals from dog olfactory tissue.
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ABSTRACT:
SUBMITTER: Dreesen TD
PROVIDER: S-EPMC1158194 | biostudies-other | 1982 Apr
REPOSITORIES: biostudies-other
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Project description:Structures of membrane proteins determined by X-ray crystallography and, increasingly, by cryo-electron microscopy often fail to resolve the structural details of unstable or reactive small molecular ligands in their physiological sites. This work demonstrates that 13C chemical shifts measured by magic-angle spinning (MAS) solid-state NMR (SSNMR) provide unique information on the conformation of a labile ligand in the physiological site of a functional protein in its native membrane, by exploiting freeze-trapping to stabilise the complex. We examine the ribose conformation of ATP in a high affinity complex with Na,K-ATPase (NKA), an enzyme that rapidly hydrolyses ATP to ADP and inorganic phosphate under physiological conditions. The 13C SSNMR spectrum of the frozen complex exhibits peaks from all ATP ribose carbon sites and some adenine base carbons. Comparison of experimental chemical shifts with density functional theory (DFT) calculations of ATP in different conformations and protein environments reveals that the ATP ribose ring adopts an C3'-endo (N) conformation when bound with high affinity to NKA in the E1Na state, in contrast to the C2'-endo (S) ribose conformations of ATP bound to the E2P state and AMPPCP in the E1 complex. Additional dipolar coupling-mediated measurements of H-C-C-H torsional angles are used to eliminate possible relative orientations of the ribose and adenine rings. The utilization of chemical shifts to determine membrane protein ligand conformations has been underexploited to date and here we demonstrate this approach to be a powerful tool for resolving the fine details of ligand-protein interactions.
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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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Project description: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.
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Project description:Na(+)/K(+) pumps move net charge through the cell membrane by mediating unequal exchange of intracellular Na(+) and extracellular K(+). Most charge moves during transitions that release Na(+) to the cell exterior. When pumps are constrained to bind and release only Na(+), a membrane voltage-step redistributes pumps among conformations with zero, one, two or three bound Na(+), thereby transiently generating current. By applying rapid voltage steps to squid giant axons, we previously identified three components in such transient currents, with distinct relaxation speeds: fast (which nearly parallels the voltage-jump time course), medium speed (?(m)=0.2-0.5?ms) and slow (?(s)=1-10?ms). Here we show that these three components are tightly correlated, both in their magnitudes and in the time courses of their changes. The correlations reveal the dynamics of the conformational rearrangements that release three Na(+) to the exterior (or sequester them into their binding sites) one at a time, in an obligatorily sequential manner.
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Project description:To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,β,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the β-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular β,β-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.
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Project description:Whole hearts from wild-type and Na,K-ATPase alpha 1 het. mice. Adult male, 8-16 weeks old on a 129/BSwiss background. Keywords: repeat sample
2004-11-19 | GSE1134 | GEO
Project description:Cyclin G-associated kinase (GAK) is a ubiquitous serine/threonine kinase that facilitates clathrin uncoating during vesicle trafficking. GAK phosphorylates a coat adaptor component, AP2M1, to help achieve this function. GAK is also implicated in Parkinson's disease through genome-wide association studies. However, GAK's role in mammalian neurons remains unclear, and insight may come from identification of further substrates. Employing a chemical genetics method, we show here that the sodium potassium pump (Na+/K+-ATPase) ?-subunit Atp1a3 is a GAK target and that GAK regulates Na+/K+-ATPase trafficking to the plasma membrane. Whole-cell patch clamp recordings from CA1 pyramidal neurons in GAK conditional knockout mice show a larger change in resting membrane potential when exposed to the Na+/K+-ATPase blocker ouabain, indicating compromised Na+/K+-ATPase function in GAK knockouts. Our results suggest a modulatory role for GAK via phosphoregulation of substrates such as Atp1a3 during cargo trafficking.
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