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Bjørn P. Pedersen - One of the best experts on this subject based on the ideXlab platform.
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serine phosphorylation regulates the p type Potassium Pump kdpfabc
eLife, 2020Co-Authors: Marie E. Sweet, Bjørn P. Pedersen, Vikas Dubey, Xihui Zhang, Thomas A. Neubert, Himanshu Khandelia, Hediye Erdjumentbromage, David L. StokesAbstract:KdpFABC is an ATP-dependent K+ Pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K+ levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the Pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type Pumps and this study furthers our understanding of how bacteria control Potassium homeostasis to maintain cell volume and osmotic potential.
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Serine Phosphorylation Regulates the P-type Potassium Pump KdpFABC
2020Co-Authors: Marie E. Sweet, Bjørn P. Pedersen, Vikas Dubey, Xihui Zhang, Hediye Erdjument-bromage, Thomas A. Neubert, Himanshu Khandelia, David L. StokesAbstract:Abstract KdpFABC is an ATP-dependent K+ Pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional regulation of kdpFABC expression is well studied, a mechanism for regulating the Pump when K+ levels are restored has not been described. Here we show that KdpFABC is inhibited by serine phosphorylation when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which is reversed by serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the Pump. Analyses of partial reactions in the transport cycle show that serine phosphorylation uncouples the Pump and blocks the cycle after formation of the catalytic aspartyl phosphate intermediate (E1~P). Molecular dynamics simulations show that serine phosphorylation affects domain dynamics that explain the uncoupling. This regulatory mechanism, unique amongst P-type Pumps, furthers our understanding of how bacteria control Potassium homeostasis to maintain cell volume and osmotic potential.
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Crystal structure of the sodium–Potassium Pump
Nature, 2007Co-Authors: J. Preben Morth, Bjørn P. Pedersen, Mads S. Toustrup-jensen, Thomas L.-m. Sørensen, Janne Petersen, Jens Peter Andersen, Bente Vilsen, Poul NissenAbstract:P-type ATPases are cation Pumps of fundamental importance for all eukaryotes and many prokaryotes. Three papers this week present structural and functional studies of key members of this superfamily. The cover shows the Na^+,K^+-Pump structure, described at 3.5 Å resolution by Morth et al ., together with J. C. Skou's original notes on his discovery of its Na^+- and K^+-dependent ATPase activity 50 years ago. The paper reveals the Potassium-bound state and hints at a voltage-dependent basis of regulation, in part through kinetic experiments similar to Skou's. Olesen et al . have obtained crystallographic snapshots of the sarcoplasmic reticulum Ca^2+-ATPase, the calcium Pump, complemented by functional studies, and a complete mechanism of calcium transport is finally presented. In plants and fungi, cellular ion homeostasis and membrane potential are powered by the plasma membrane H^+-ATPase — another P-type ATPase. Pedersen et al . present its X-ray structure and provide insight into how it Pumps protons against a steep electrochemical gradient. Relatively little is known about the mechanisms that underlie the active transport of ions by Na^+,K^+-ATPase. A 3.5 Å X-ray structure of this fundamental protein is presented, revealing the two binding sites for Potassium. The Na^+,K^+-ATPase generates electrochemical gradients for sodium and Potassium that are vital to animal cells, exchanging three sodium ions for two Potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 Å resolution of the pig renal Na^+,K^+-ATPase with two rubidium ions bound (as Potassium congeners) in an occluded state in the transmembrane part of the α-subunit. Several of the residues forming the cavity for rubidium/Potassium occlusion in the Na^+,K^+-ATPase are homologous to those binding calcium in the Ca^2+-ATPase of sarco(endo)plasmic reticulum. The β- and γ-subunits specific to the Na^+,K^+-ATPase are associated with transmembrane helices αM7/αM10 and αM9, respectively. The γ-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the α-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.
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crystal structure of the sodium Potassium Pump
Nature, 2007Co-Authors: Bjørn P. Pedersen, Janne Petersen, Preben J Morth, Mads S Toustrupjensen, Thomas Sorensen, Jens Peter AndersenAbstract:The Na+,K+-ATPase generates electrochemical gradients for sodium and Potassium that are vital to animal cells, exchanging three sodium ions for two Potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 A resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as Potassium congeners) in an occluded state in the transmembrane part of the α-subunit. Several of the residues forming the cavity for rubidium/Potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The β- and γ-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices αM7/αM10 and αM9, respectively. The γ-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the α-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential. P-type ATPases are cation Pumps of fundamental importance for all eukaryotes and many prokaryotes. Three papers this week present structural and functional studies of key members of this superfamily. The cover shows the Na+,K+-Pump structure, described at 3.5 A resolution by Morth et al., together with J. C. Skou's original notes on his discovery of its Na+- and K+-dependent ATPase activity 50 years ago. The paper reveals the Potassium-bound state and hints at a voltage-dependent basis of regulation, in part through kinetic experiments similar to Skou's. Olesen et al. have obtained crystallographic snapshots of the sarcoplasmic reticulum Ca2+-ATPase, the calcium Pump, complemented by functional studies, and a complete mechanism of calcium transport is finally presented. In plants and fungi, cellular ion homeostasis and membrane potential are powered by the plasma membrane H+-ATPase — another P-type ATPase. Pedersen et al. present its X-ray structure and provide insight into how it Pumps protons against a steep electrochemical gradient. Relatively little is known about the mechanisms that underlie the active transport of ions by Na+,K+-ATPase. A 3.5 A X-ray structure of this fundamental protein is presented, revealing the two binding sites for Potassium.
Chikashi Toyoshima - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the sodium Potassium Pump na k atpase with bound Potassium and ouabain
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Haruo Ogawa, Takehiro Shinoda, Flemming Cornelius, Chikashi ToyoshimaAbstract:The sodium-Potassium Pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.
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Crystal structure of the sodium-Potassium Pump (Na+,K+-ATPase) with bound Potassium and ouabain
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Haruo Ogawa, Takehiro Shinoda, Flemming Cornelius, Chikashi ToyoshimaAbstract:The sodium-Potassium Pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.
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Crystal structure of the sodium-Potassium Pump at 2.4 resolution
Nature, 2009Co-Authors: Takehiro Shinoda, Haruo Ogawa, Flemming Cornelius, Chikashi ToyoshimaAbstract:Sodium-Potassium ATPase is an ATP-powered ion Pump that establishes concentration gradients for Na(+) and K(+) ions across the plasma membrane in all animal cells by Pumping Na(+) from the cytoplasm and K(+) from the extracellular medium. Such gradients are used in many essential processes, notably for generating action potentials. Na(+), K(+)-ATPase is a member of the P-type ATPases, which include sarcoplasmic reticulum Ca(2+)-ATPase and gastric H(+), K(+)-ATPase, among others, and is the target of cardiac glycosides. Here we describe a crystal structure of this important ion Pump, from shark rectal glands, consisting of alpha- and beta-subunits and a regulatory FXYD protein, all of which are highly homologous to human ones. The ATPase was fixed in a state analogous to E2.2K(+).P(i), in which the ATPase has a high affinity for K(+) and still binds P(i), as in the first crystal structure of pig kidney enzyme at 3.5 A resolution. Clearly visualized now at 2.4 A resolution are coordination of K(+) and associated water molecules in the transmembrane binding sites and a phosphate analogue (MgF(4)(2-)) in the phosphorylation site. The crystal structure shows that the beta-subunit has a critical role in K(+) binding (although its involvement has previously been suggested) and explains, at least partially, why the homologous Ca(2+)-ATPase counter-transports H(+) rather than K(+), despite the coordinating residues being almost identical.
David L. Stokes - One of the best experts on this subject based on the ideXlab platform.
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serine phosphorylation regulates the p type Potassium Pump kdpfabc
eLife, 2020Co-Authors: Marie E. Sweet, Bjørn P. Pedersen, Vikas Dubey, Xihui Zhang, Thomas A. Neubert, Himanshu Khandelia, Hediye Erdjumentbromage, David L. StokesAbstract:KdpFABC is an ATP-dependent K+ Pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K+ levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the Pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type Pumps and this study furthers our understanding of how bacteria control Potassium homeostasis to maintain cell volume and osmotic potential.
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Serine Phosphorylation Regulates the P-type Potassium Pump KdpFABC
2020Co-Authors: Marie E. Sweet, Bjørn P. Pedersen, Vikas Dubey, Xihui Zhang, Hediye Erdjument-bromage, Thomas A. Neubert, Himanshu Khandelia, David L. StokesAbstract:Abstract KdpFABC is an ATP-dependent K+ Pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional regulation of kdpFABC expression is well studied, a mechanism for regulating the Pump when K+ levels are restored has not been described. Here we show that KdpFABC is inhibited by serine phosphorylation when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which is reversed by serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the Pump. Analyses of partial reactions in the transport cycle show that serine phosphorylation uncouples the Pump and blocks the cycle after formation of the catalytic aspartyl phosphate intermediate (E1~P). Molecular dynamics simulations show that serine phosphorylation affects domain dynamics that explain the uncoupling. This regulatory mechanism, unique amongst P-type Pumps, furthers our understanding of how bacteria control Potassium homeostasis to maintain cell volume and osmotic potential.
Jolanta Chodakowska - One of the best experts on this subject based on the ideXlab platform.
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Effect of hemodialysis on the level of serum digoxin-like substance and sodium-Potassium Pump activity in erythrocytes from patients with chronic renal failure
Polskie Archiwum Medycyny Wewnetrznej, 1992Co-Authors: Czarkowski M, Jabłońska-skwiecińska E, Drygieniec D, Wardyn K, Rojek-trebicka J, Ignatowska-switalska H, Staniszewska K, Jolanta ChodakowskaAbstract:Digoxin-like immunoreactivity (DLS) and erythrocyte sodium-Potassium Pump (PSP) activity were measured in a group of 16 patients with chronic renal failure (CRF) before and just after haemodialysis and in a group of 9 healthy persons. Before haemodialysis DLS was present in the blood of most CRF patients, at the mean concentration of 0,14 +/- 0,13 micrograms/l. After haemodialysis DLS concentration decreased to 0,09 +/- 0,09 microgram/l (p less than 0,01). In the control group blood DLS concentration was nondetectable. In the CRF group PSP activity was higher before than after haemodialysis (p less than 0,01; 12,1 +/- 1,8 and 7,6 +/- 1,4 muMol Pi/h/g Hb. PSP activity in the control groups was 10,3 +/- 1,9 muMol Pi/h/g Hb). In the CRF group PSP activity was higher before haemodialysis (p less than 0,05) and lower after haemodialysis (p less than 0.01) than in the control group. Our results confirmed the presence of DLS in the blood of the majority of CRF patients. DLS concentration decreased after haemodialysis but we did not found any parallel increase in PSP activity in these patients. These results did not confirm the hypothesis that DLS might inhibit PSP activity in red blood cells from CRF patients.
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Activity of the sodium-Potassium Pump and values of sodium and Potassium in erythrocytes in patients with non-dialyzed renal failure and arterial hypertension treated conservatively
Polskie Archiwum Medycyny Wewnetrznej, 1991Co-Authors: Czarkowski M, Jabłońska-skwiecińska E, Drygieniec D, Wardyn K, Rojek-trebicka J, Jolanta ChodakowskaAbstract:In 8 non-dialysed patients with chronic renal failure (PNN) and hypertension (NT)-(PNNT group) sodium-Potassium Pump activity (PSP) and intra-erythrocyte sodium (NaE) and Potassium (KE) concentration were measured. No differences were found in PSP, NaE and KE between group PNNT and healthy volunteers (Z). These results do not support an importance of the role of the so-called endogenous PSP inhibitor in the pathogenesis of NT in patients with PNN.
Antonio-carlos G. De Almeida - One of the best experts on this subject based on the ideXlab platform.
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Palytoxin and the sodium/Potassium Pump--phosphorylation and Potassium interaction.
Physical biology, 2009Co-Authors: Antônio M. Rodrigues, Antonio Fernando Catelli Infantosi, Antonio-carlos G. De AlmeidaAbstract:We proposed a reaction model for investigating interactions between K+ and the palytoxin-sodium-Potassium (PTX-Na+/K+) Pump complex under conditions where enzyme phosphorylation may occur. The model is composed of (i) the Albers-Post model for Na+/K+-ATPase, describing Na+ and K+ Pumping; (ii) the reaction model proposed for Na+/K+-ATPase interactions with its ligands (Na+, K+, ATP, ADP and P) and with PTX. A mathematical model derived for representing the reactions was used to simulate experimental studies of the PTX-induced current, in different concentrations for the Pump ligands. The simulations allow interpretation of the simultaneous action of Na+/K+-ATPase phosphorylation and K+ on the PTX-induced channels. The results suggest that(i) phosphorylation increases the PTX toxic effect, increasing its affinity and reducing the K+occlusion rate, and (ii) K+ causes channel blockage, increases the toxin dissociation rate and impedes the induced channel phosphorylation, implying reduction of the PTX toxic effect.
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palytoxin and the sodium Potassium Pump phosphorylation and Potassium interaction
Physical Biology, 2009Co-Authors: Antônio M. Rodrigues, Antonio Fernando Catelli Infantosi, Antonio-carlos G. De AlmeidaAbstract:We proposed a reaction model for investigating interactions between K+ and the palytoxin-sodium-Potassium (PTX-Na+/K+) Pump complex under conditions where enzyme phosphorylation may occur. The model is composed of (i) the Albers-Post model for Na+/K+-ATPase, describing Na+ and K+ Pumping; (ii) the reaction model proposed for Na+/K+-ATPase interactions with its ligands (Na+, K+, ATP, ADP and P) and with PTX. A mathematical model derived for representing the reactions was used to simulate experimental studies of the PTX-induced current, in different concentrations for the Pump ligands. The simulations allow interpretation of the simultaneous action of Na+/K+-ATPase phosphorylation and K+ on the PTX-induced channels. The results suggest that(i) phosphorylation increases the PTX toxic effect, increasing its affinity and reducing the K+occlusion rate, and (ii) K+ causes channel blockage, increases the toxin dissociation rate and impedes the induced channel phosphorylation, implying reduction of the PTX toxic effect.
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Effect of palytoxin on the sodium-Potassium Pump: model and simulation.
Physical biology, 2008Co-Authors: Antônio M. Rodrigues, Antonio-carlos G. De Almeida, Antonio Fernando Catelli InfantosiAbstract:We propose a reaction model for the palytoxin-sodium-Potassium (PTX-Na(+)/K(+)) Pump complex. The model, which is similar to the Albers-Post model for Na(+)/K(+)-ATPase, is used to elucidate the effect of PTX on Na(+)/K(+)-ATPase during the enzyme interactions with Na(+) and/or K(+) ions. Conformational substates and reactions for the Pump are incorporated into the Albers-Post model to represent enzymes with or without bound PTX. A mathematical model based on the reaction scheme is used in simulations modeling experimental studies of PTX-induced ionic currents. Our simulations suggest that (i) extracellular Na(+) as well as K(+) promotes PTX-induced channel blockage; (ii) extracellular K(+) accelerates PTX unbinding; and (iii) K(+) occlusion in the PTX-Pump complex is essential for describing the PTX-induced current dynamics.