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:Phosphatidylserine (PtdSer) incubated with synaptosomal plasma membranes (SPM) of dog brain is incorporated into SPM in proportion to its concentration in the incubation medium. Low PtdSer concentrations progressively activated the SPM-associated (Na+ + K+)-stimulated ATPase and acetylcholinesterase. Increasing the PtdSer concentration above that which maximally stimulated the enzyme activities effected a progressive inhibition with respect to maximal stimulation. Arrhenius plots of (Na+ + K+ + Mg2+)-dependent ATPase and 5'-nucleotidase revealed a clear break at 23-24 degrees C for both enzymes in SPM untreated with PtdSer (controls), whereas a linear relation was obtained for SPM treated with PtdSer. Changes in the allosteric properties of (Na+ + K+)-stimulated ATPase by fluoride (F-) and/or of 5'-nucleotidase by concanavalin A (i.e. changes of Hill coefficients) indicate that PtdSer increases the membrane fluidity. These results suggest that modifications of lipid-protein interactions in SPM induced by PtdSer may have implications in the physiological processes in the central nervous system.
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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.
| S-EPMC6463725 | biostudies-literature
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.
| S-EPMC3293407 | biostudies-literature
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.
| S-EPMC3186040 | biostudies-other
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:Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K(+). Studies with purified membranes revealed that CPZ reduced Na(+)-dependent phosphorylation by interference with Na(+) binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K(+). Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive (22)Na(+) influx into K(+)-loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na(+) cycle from an Na(+)/K(+) cycle in the pump. The Na(+) cycle possibly involves transport through the recently characterized Na(+)-specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na(+) efflux by modifying the transport stoichiometry of single pump units.
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Project description:Over the last decades several efforts have been carried out to determine the mechanisms of salt homeostasis in plants and, more recently, to identify genes implicated in salt tolerance, with some plants being successfully genetically engineered to improve resistance to salt. It is well established that the efficient exclusion of Na(+) excess from the cytoplasm and vacuolar Na(+) accumulation are the most important steps towards the maintenance of ion homeostasis inside the cell. Therefore, the vacuole of plant cells plays a pivotal role in the storage of salt. After the identification of the vacuolar Na(+)/H(+) antiporter Nhx1 in Saccharomyces cerevisiae, the first plant Na(+)/H(+) antiporter, AtNHX1, was isolated from Arabidopsis and its overexpression resulted in plants exhibiting increased salt tolerance. Also, the identification of the plasma membrane Na(+)/H(+) exchanger SOS1 and how it is regulated by a protein kinase SOS2 and a calcium binding protein SOS3 were great achievements in the understanding of plant salt resistance. Both tonoplast and plasma membrane antiporters exclude Na+ from the cytosol driven by the proton-motive force generated by the plasma membrane H(+)-ATPase and by the vacuolar membrane H(+)-ATPase and H(+)-pyrophosphatase and it has been shown that the activity of these proteins responds to salinity. In this review we focus on the transcriptional and post-transcriptional regulation by salt of tonoplast proton pumps and Na(+)/H(+) exchangers and on the signalling pathways involved in salt sensing.
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Project description:The protein-protein interactions amongst the Na/K-ATPase α1 subunit, c-Src, and caveolin-1 (cav-1) are essential for the Na/K-ATPase signaling functions. However, there are arguments concerning the interaction model. The present study aims to clarify the interactions amongst the endogenous native proteins in live cells under native resting condition. Under native condition, Blue Native-PAGE and Blue Native-PAGE/SDS-PAGE 2D analyses demonstrated co-existence of the α1 subunit and c-Src in same protein complex, as well as a direct interaction between the α1 subunit and c-Src. By comparison of cleavable and non-cleavable cysteine-cysteine crosslinked samples, capillary immunoblotting analysis demonstrated that depletion of Src kinase family members (c-Src, Yes, and Fyn) or cav-1 clearly reduced the interactions of the α1 subunit with proteins, but depletion of cav-1 did not affect the interaction of c-Src with the α1 subunit. The data indicated that there are direct interactions between the α1 subunit and c-Src as well as between the α1 subunit and cav-1, but argued about the interaction between c-Src and cav-1 under the condition. Furthermore, the data also indicated the existence of different protein complexes containing the α1 subunit and c-Src, which might have different signaling functions.
| S-EPMC7138855 | biostudies-literature
Project description:BackgroundDogs were an important element in many native American cultures at the time Europeans arrived. Although previous ancient DNA studies revealed the existence of unique native American mitochondrial sequences, these have not been found in modern dogs, mainly purebred, studied so far.ResultsWe identified many previously undescribed mitochondrial control region sequences in 400 dogs from rural and isolated areas as well as street dogs from across the Americas. However, sequences of native American origin proved to be exceedingly rare, and we estimate that the native population contributed only a minor fraction of the gene pool that constitutes the modern population.ConclusionsThe high number of previously unidentified haplotypes in our sample suggests that a lot of unsampled genetic variation exists in non-breed dogs. Our results also suggest that the arrival of European colonists to the Americas may have led to an extensive replacement of the native American dog population by the dogs of the invaders.
| S-EPMC3076259 | biostudies-literature