Intrinsic reaction-cycle time scale of Na+,K+-ATPase manifests itself in the lipid-protein interactions of nonequilibrium membranes.
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ABSTRACT:
SUBMITTER: Bouvrais H
PROVIDER: S-EPMC3494963 | biostudies-other | 2012 Nov
REPOSITORIES: biostudies-other
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Project description:The whole-cell voltage-clamp technique was used in rat cardiac myocytes to investigate the kinetics of ADP binding to phosphorylated states of Na,K-ATPase and its effects on presteady-state Na(+)-dependent charge movements by this enzyme. Ouabain-sensitive transient currents generated by Na,K-ATPase functioning in electroneutral Na(+)-Na(+) exchange mode were measured at 23 degrees C with pipette ADP concentrations ([ADP]) of up to 4.3 mM and extracellular Na(+) concentrations ([Na](o)) between 36 and 145 mM at membrane potentials (V(M)) from -160 to +80 mV. Analysis of charge-V(M) curves showed that the midpoint potential of charge distribution was shifted toward more positive V(M) both by increasing [ADP] at constant Na(+)(o) and by increasing [Na](o) at constant ADP. The total quantity of mobile charge, on the other hand, was found to be independent of changes in [ADP] or [Na](o). The presence of ADP increased the apparent rate constant for current relaxation at hyperpolarizing V(M) but decreased it at depolarizing V(M) as compared to control (no added ADP), an indication that ADP binding facilitates backward reaction steps during Na(+)-Na(+) exchange while slowing forward reactions. Data analysis using a pseudo three-state model yielded an apparent K(d) of approximately 6 mM for ADP binding to and release from the Na,K-ATPase phosphoenzyme; a value of 130 s(-1) for k(2), a rate constant that groups Na(+) deocclusion/release and the enzyme conformational transition E(1) approximately P --> E(2)-P; a value of 162 s(-1)M(-1) for k(-2), a lumped second-order V(M)-independent rate constant describing the reverse reactions; and a Hill coefficient of approximately 1 for Na(+)(o) binding to E(2)-P. The results are consistent with electroneutral release of ADP before Na(+) is deoccluded and released through an ion well. The same approach can be used to study additional charge-moving reactions and associated electrically silent steps of the Na,K-pump and other transporters.
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Project description:The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from (2)H-hyperfine interaction with D2O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D2O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D2O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D2O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D2O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.
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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
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Project description:The multivesicular body (MVB) pathway functions in multiple cellular processes including cell surface receptor down-regulation and viral budding from host cells. An important step in the MVB pathway is the correct sorting of cargo molecules, which requires the assembly and disassembly of endosomal sorting complexes required for transport (ESCRTs) on the endosomal membrane. Disassembly of the ESCRTs is catalyzed by ATPase associated with various cellular activities (AAA) protein Vps4. Vps4 contains a single AAA domain and undergoes ATP-dependent quaternary structural change to disassemble the ESCRTs. Structural and biochemical analyses of the Vps4 ATPase reaction cycle are reported here. Crystal structures of Saccharomyces cerevisiae Vps4 in both the nucleotide-free form and the ADP-bound form provide the first structural view illustrating how nucleotide binding might induce conformational changes within Vps4 that lead to oligomerization and binding to its substrate ESCRT-III subunits. In contrast to previous models, characterization of the Vps4 structure now supports a model where the ground state of Vps4 in the ATPase reaction cycle is predominantly a monomer and the activated state is a dodecamer. Comparison with a previously reported human VPS4B structure suggests that Vps4 functions in the MVB pathway via a highly conserved mechanism supported by similar protein-protein interactions during its ATPase reaction cycle.
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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:Early afterdepolarizations (EADs) are triggers of cardiac arrhythmia driven by L-type Ca(2+) current (ICaL) reactivation or sarcoplasmic reticulum Ca(2+) release and Na(+)/Ca(2+) exchange. In large mammals the positive action potential plateau promotes ICaL reactivation, and the current paradigm holds that cardiac EAD dynamics are dominated by interaction between ICaL and the repolarizing K(+) currents. However, EADs are also frequent in the rapidly repolarizing mouse action potential, which should not readily permit ICaL reactivation. This suggests that murine EADs exhibit unique dynamics, which are key for interpreting arrhythmia mechanisms in this ubiquitous model organism. We investigated these dynamics in myocytes from arrhythmia-susceptible calcium calmodulin-dependent protein kinase II delta C (CaMKII?C)-overexpressing mice (Tg), and via computational simulations.In Tg myocytes, ?-adrenergic challenge slowed late repolarization, potentiated sarcoplasmic reticulum Ca(2+) release, and initiated EADs below the ICaL activation range (-47 ± 0.7 mV). These EADs were abolished by caffeine and tetrodotoxin (but not ranolazine), suggesting that sarcoplasmic reticulum Ca(2+) release and Na(+) current (INa), but not late INa, are required for EAD initiation. Simulations suggest that potentiated sarcoplasmic reticulum Ca(2+) release and Na(+)/Ca(2+) exchange shape late action potential repolarization to favor nonequilibrium reactivation of INa and thereby drive the EAD upstroke. Action potential clamp experiments suggest that lidocaine eliminates virtually all inward current elicited by EADs, and that this effect occurs at concentrations (40-60 ?mol/L) for which lidocaine remains specific for inactivated Na(+) channels. This strongly suggests that previously inactive channels are recruited during the EAD upstroke, and that nonequilibrium INa dynamics underlie murine EADs.Nonequilibrium reactivation of INa drives murine EADs.
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Project description:The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na+-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km for Na+ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na+ efflux to increase in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.
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Project description:Rat colonic basolateral membranes were incubated with S-adenosyl-L-[methyl-3H]methionine (0.3 mM) at 37 degrees C for 2 h at pH 9.0. This resulted in an increase in the specific activity of Na+ + K+-ATPase by 60%. Kinetic parameter analysis revealed a 2-fold increase in the Vmax. of this enzymatic activity, whereas the Km for ATP was unchanged. The methylation inhibitor S-adenosyl-L-homocysteine (2 mM) significantly reduced these S-adenosyl-L-methionine-stimulated increases in specific activity and the Vmax. of Na+ + K+-ATPase. S-Adenosyl-L-methionine treatment of basolateral membranes was also found to significantly increase the fluidity of these preparations, as assessed by steady-state fluorescence polarization techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene; S-adenosyl-L-homocysteine (2 mM) again markedly reduced this S-adenosyl-L-methionine-induced increase in fluidity. While transmethylation reactions involving phospholipids, non-polar lipids and proteins were all found to exist in rat colonic basolateral membranes, based on a number of observations, the results of the present studies suggest that transmethylation of membrane phospholipids, but not membrane non-polar lipids or proteins, influenced the fluidity of basolateral membranes which, in turn, modified Na+ + K+-ATPase activity in these membranes.
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