Structure-function studies of the sodium pump

1999 ◽  
Vol 77 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rhoda Blostein
Keyword(s):  
Sodium Pump ◽  
Atp Hydrolysis ◽  
Cation Binding ◽  
Structural Basis ◽  
Animal Cells ◽  
Sodium Potassium ◽  
Membrane Protein Complex ◽  
And Function ◽  
P Type ◽  
Hydrolysis Of

The Na+,K+-ATPase is an ubiquitous plasma membrane protein complex that belongs to the P-type family of ion motive ATPases. Under normal conditons, it couples the hydrolysis of one molecule of ATP to the exchange of three Na+ for two K+ ions, thus maintaining the normal gradient of these cations in animal cells. Despite decades of investigation of its structure and function, the structural basis for its cation specificity and for conformational coupling of the scalar energy of ATP hydrolysis to the vectorial movement of Na+ and K+ have remained a major unresolved issue. This paper summarizes our recent studies concerned with these issues. The findings indicate that regions(s) of the amino terminus and first cytoplasmic (M2/M3) loop act synergisticaly to affect the steady-state conformational equilibrium of the enzyme. Although carboxyl- or hydroxyl-bearing amino acids comprise the cation-binding and occlusion sites, our experiments also suggest that these interactions may be modulated by juxtapositioned cytoplasmic regions.Key words: sodium, potassium, ATPase, Na+,K+-ATPase, sodium pump.

scholarly journals A single K+-binding site in the crystal structure of the gastric proton pump

2019 ◽  
Author(s):  
Kenta Yamamoto ◽  
Vikas Dubey ◽  
Katsumasa Irie ◽  
Hanayo Nakanishi ◽  
Himanshu Khandelia ◽  
...  
Keyword(s):  
Binding Site ◽  
Proton Pump ◽  
Atp Hydrolysis ◽  
Energy Requirement ◽  
Cation Binding ◽  
Structural Basis ◽  
Cation Binding Site ◽  
Gastric Proton Pump ◽  
Dynamics Simulations ◽  
P Type

AbstractThe gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold – the highest cation gradient known in any mammalian tissue – across the membrane.

Biogenesis and function of the yeast plasma-membrane H(+)-ATPase

2000 ◽  
Vol 203 (1) ◽  
pp. 155-160 ◽  
Author(s):  
A. Ambesi ◽  
M. Miranda ◽  
V.V. Petrov ◽  
C.W. Slayman
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Proton Pumping ◽  
Site Directed Mutagenesis ◽  
Animal Cells ◽  
Alpha Helices ◽  
Yeast Plasma Membrane ◽  
High Affinity ◽  
And Function ◽  
P Type

One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters. The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices. It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate. Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution. The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites. In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis. This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping. Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling.

scholarly journals A single K+-binding site in the crystal structure of the gastric proton pump

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Kenta Yamamoto ◽  
Vikas Dubey ◽  
Katsumasa Irie ◽  
Hanayo Nakanishi ◽  
Himanshu Khandelia ◽  
...  
Keyword(s):  
Binding Site ◽  
Proton Pump ◽  
Atp Hydrolysis ◽  
Energy Requirement ◽  
Cation Binding ◽  
Structural Basis ◽  
Cation Binding Site ◽  
Gastric Proton Pump ◽  
Dynamics Simulations ◽  
P Type

The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of the H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfills the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved and supported by molecular dynamics simulations, establishing how the H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold—one of the highest cation gradients known in mammalian tissue—across the membrane.

The sodium pump in the evolution of animal cells

1995 ◽  
Vol 349 (1329) ◽  
pp. 263-269 ◽  
Keyword(s):  
Sodium Pump ◽  
Transport Systems ◽  
Primary Role ◽  
Bacterial Cells ◽  
Electrochemical Gradient ◽  
Animal Cells ◽  
Osmotic Balance ◽  
Energy Consuming ◽  
P Type

Plant cells and bacterial cells are surrounded by a massive cellulose wall, which constrains their high internal osmotic pressure (tens of atmospheres). Animal cells, in contrast, are in osmotic equilibrium with their environment, have no restraining surround, can take on a variety of shapes and change these from moment to moment. This osmotic balance is achieved by the action of the energy-consuming sodium pump, one of the P-type ATPase transport protein family, members of which are indeed also found in bacteria. The pump’s action brings about a transmembranal electrochemical gradient of sodium ions, harnessed in a range of transport systems that couple the dissipation of this gradient to establishing a gradient of the coupled substrate. The primary role of the sodium pump as a regulator of cell volume has evolved to provide the basis for an enormous variety of physiological functions.

Cryo-EM structures of the human sodium-potassium pump revealing the gating mechanism on the cytoplasmic side

2021 ◽  
Author(s):  
Yingying Guo ◽  
Yuanyuan Zhang ◽  
Renhong Yan ◽  
Bangdong Huang ◽  
Fangfei Ye ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Structural Basis ◽  
Extracellular Potassium ◽  
Ion Pump ◽  
Sodium Potassium ◽  
Working Cycle ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Membrane Bound ◽  
Cytoplasmic Side

Abstract Na+/K+-ATPase (NKA) is a membrane-bound ion pump that generates electrochemical gradient of sodium ion and potassium ion across the plasma membrane via hydrolyzing ATP. During each so-called Post-Albers cycle, NKA exchanges three cytoplasmic sodium ions for two extracellular potassium ions through alternating E1 and E2 states. Hitherto, there are several steps remained unknown during the complete working cycle of NKA. Here, we report cryo-electron microscopy (cryo-EM) structures of recombinant over-expressed human NKA in three distinct states at 3.1–3.4 Å resolution, representing the E1·3Na state, in which the cytosolic gate is open, and the E1·3Na·ATP state preceding ATP hydrolysis and a basic E2·[2K] state. These structures reveal the ATP-dependent Na+-binding site remodeling for the close of the cytoplasmic gate, filling a gap in the structural elucidation of the Post-Albers cycle of NKA and providing structural basis for understanding the cytoplasmic Na+ entrance pathway.

Inhibition of the Ecto-ATPdiphosphohydrolase of Schistosoma mansoni by Thapsigargin

2000 ◽  
Vol 20 (5) ◽  
pp. 369-381 ◽  
Author(s):  
Samantha M. Martins ◽  
Christiane R. Torres ◽  
Sérgio T. Ferreira
Keyword(s):  
Schistosoma Mansoni ◽  
Active Site ◽  
Calcium Transport ◽  
Atp Hydrolysis ◽  
External Surface ◽  
Specific Inhibitor ◽  
Etiologic Agent ◽  
Endoplasmic Reticulum Calcium ◽  
P Type ◽  
Hydrolysis Of

ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable ofhydrolyzing nucleoside di- and triphosphates. Although a number ofpossible physiological roles have been proposed for ATPDases, detailedstudies on structure-function relationships have generally been hamperedby the lack of specific inhibitors of these enzymes. We have previouslycharacterized a Ca2+-activated ATPDase on the external surface ofthe tegument of Schistosoma mansoni, the etiologic agent of humanschistosomiasis. In the present work, we have examined the effectsof thapsigargin, a sesquiterpene lactone known as a high affinityinhibitor of sarco-endoplasmic reticulum calcium transport (SERCA)ATPase, on ATPDase activity. Whereas other lactones tested had littleor no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (Ki∼20 μM). Interestingly, hydrolysis of ADP was notinhibited by thapsigargin. The lack of inhibition of ATPase activityby orthovanadate, a specific inhibitor of P-type ATPases, and theinhibition of the Mg2+-stimulated ATP hydrolysis by thapsigarginruled out the possibility that the observed inhibition of the ATPDaseby thapsigargin could be due to the presence of contaminating SERCAATPases in our preparation. Kinetic analysis indicated that a singleactive site in the ATPDase is responsible for hydrolysis of both ATPand ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigarginwas little or not affected by changes in free Ca2+ or Mg2+concentrations. These results suggest that interaction of thapsigarginwith the S. mansoni ATPDase prevents binding of ATP or its hydrolysisat the active site, while ADP can still undergo catalysis.

scholarly journals Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase

2016 ◽  
Vol 114 (2) ◽  
pp. 316-321 ◽  
Author(s):  
Rikke Holm ◽  
Jaanki Khandelwal ◽  
Anja P. Einholm ◽  
Jens P. Andersen ◽  
Pablo Artigas ◽  
...  
Keyword(s):  
Turnover Rate ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Cation Binding ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Wild Type ◽  
Transport Mode ◽  
The Third ◽  
Enzyme Turnover

Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type–like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

The sarcoplasmic Ca2+-ATPase: design of a perfect chemi-osmotic pump

2010 ◽  
Vol 43 (4) ◽  
pp. 501-566 ◽  
Author(s):  
Jesper V. Møller ◽  
Claus Olesen ◽  
Anne-Marie L. Winther ◽  
Poul Nissen
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transport Mechanism ◽  
Atp Hydrolysis ◽  
Cation Binding ◽  
Thermodynamic Efficiency ◽  
Lipid Phase ◽  
Conformational States ◽  
Sarcoplasmic Reticulum Membrane ◽  
Cytosolic Domains ◽  
P Type

AbstractThe sarcoplasmic (SERCA 1a) Ca2+-ATPase is a membrane protein abundantly present in skeletal mucles where it functions as an indispensable component of the excitation–contraction coupling, being at the expense of ATP hydrolysis involved in Ca2+/H+ exchange with a high thermodynamic efficiency across the sarcoplasmic reticulum membrane. The transporter serves as a prototype of a whole family of cation transporters, the P-type ATPases, which in addition to Ca2+ transporting proteins count Na+, K+-ATPase and H+, K+-, proton- and heavy metal transporting ATPases as prominent members. The ability in recent years to produce and analyze at atomic (2·3–3 Å) resolution 3D-crystals of Ca2+-transport intermediates of SERCA 1a has meant a breakthrough in our understanding of the structural aspects of the transport mechanism. We describe here the detailed construction of the ATPase in terms of one membraneous and three cytosolic domains held together by a central core that mediates coupling between Ca2+-transport and ATP hydrolysis. During turnover, the pump is present in two different conformational states, E1 and E2, with a preference for the binding of Ca2+ and H+, respectively. We discuss how phosphorylated and non-phosphorylated forms of these conformational states with cytosolic, occluded or luminally exposed cation-binding sites are able to convert the chemical energy derived from ATP hydrolysis into an electrochemical gradient of Ca2+ across the sarcoplasmic reticulum membrane. In conjunction with these basic reactions which serve as a structural framework for the transport function of other P-type ATPases as well, we also review the role of the lipid phase and the regulatory and thermodynamic aspects of the transport mechanism.

scholarly journals Structural basis for potassium transport in prokaryotes by KdpFABC

2021 ◽  
Vol 118 (29) ◽  
pp. e2105195118
Author(s):  
Marie E. Sweet ◽  
Casper Larsen ◽  
Xihui Zhang ◽  
Michael Schlame ◽  
Bjørn P. Pedersen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Structural Basis ◽  
Membrane Domain ◽  
Ionic Homeostasis ◽  
Substrate Analogs ◽  
Structural Work ◽  
P Type ◽  
Alternative Sites ◽  
Transport Complex

KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K+ transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.

Role of Sarco/Endoplasmic Reticulum Ca2+-ATPase in Thermogenesis

2005 ◽  
Vol 25 (3-4) ◽  
pp. 181-190 ◽  
Author(s):  
Leopoldo de Meis ◽  
Ana Paula Arruda ◽  
Denise P. Carvalho
Keyword(s):  
Endoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Surrounding Medium ◽  
Platelet Activating Factor ◽  
Chemical Energy ◽  
Membrane Bound ◽  
And Function ◽  
Hydrolysis Of ◽  
Heat Released

Enzymes are able to handle the energy derived from the hydrolysis of phosphate compounds in such a way as to determine the parcel that is used for work and the fraction that is converted into heat. The sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) is a family of membrane-bound ATPases that are able to transport Ca2+ ion across the membrane using the chemical energy derived from ATP hydrolysis. The heat released during ATP hydrolysis by SERCA may vary from 10 up to 30 kcal/mol depending on the SERCA isoform used and on whether or not a Ca2+ gradient is formed across the membrane. Drugs such as heparin, dimethyl sulfoxide and the platelet-activating factor (PAF) are able to modify the fraction of the chemical energy released during ATP hydrolysis that is used for Ca2+ transport and the fraction that is dissipated in the surrounding medium as heat. The thyroid hormone 3,5,3′-triiodo l-thyronine (T3) regulates the expression and function of the thermogenic SERCA isoforms. Modulation of heat production by SERCA might be one of the mechanisms involved in the increased thermogenesis found in hyperthyroidism.

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