Proton-activated rubidium transport catalyzed by the sodium pump

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.     

Principle of parametric ion separation in a molecular model of the sodium pump

Expressions were obtained for separation coefficients of sodium and potassium ions in macroscopic and molecular models of the sodium pump. Conjugation between the energy-donor process at ATP hydrolysis and ion transport is achieved at the expense of synchroneous changes of the affinity of ion-binding centers and divided cations. i. e. the cyclic changes of energetic profiles in the membrane for each type of ions. The division coefficient in the stationary state is equal to the product of relative changes in selectivity towards the cations of the enzyme ion-binding centers in phosporylated and unphosphorylated states.     

Osmotic regulation of the sodium pump in rat brain synaptosomes]

Effect of medium osmolarity on 3H-ouabain binding and the rate of ouabain-sensitive 86Rb+ transport in the rat brain synaptosomes was studied. A decrease in tonicity to 230 mOsm increases both parameters indicating the activation of the sodium pump upon synaptosome swelling. The effect is retained in the absence of inside-oriented Na+ gradient, i. e. a rise in Na(in) is not responsible for hypoosmotic activation. Colchicine (5mM) decreased and cytochalasin B (40 microM) increased the ouabain binding. In the presence of cytochalasin B the inhibition of binding observed under hypotonic conditions was shifted to higher osmolarity values. It is suggested that volume regulation of the sodium pump is controlled by the cytoskeleton elements.     

Ratio of Na:K transport in reconstituted sodium pump vesicles

The effect of ATP on the kinetics of Na and K fluxes across the membranes of reconstituted sodium pump vesicles was examined. In the absence of ATP, the active vesicles equilibrated with ⁴²K or ⁸⁶Rb within 6 hours. In contrast, the equilibration of intravesicular Na with external ²²Na was about 4 times slower. In the presence of ATP, the intravesicular K was replaced within 3 min by Na $\text{via}$ a Na:K exchange process. The total intravesicular Na pool was then labeled to the same specific radioactivity as the Na of the medium $\text{via}$ a Na:Na exchange process. The Na:K transport ratio varied with the intravesicular concentrations of Na and K.     

A voltage-activated cation transport pathway associated with the sodium pump.

In proteoliposomes containing reconstituted shark Na,K-ATPase, inside positive potentials open a cation conductance characterized by a voltage-dependence very similar to that found in mammalian erythrocytes. In both proteoliposomes and erythrocytes, the voltage-activated pathway is inhibited by external oligomycin, which traps the Na,K-ATPase in a Na-occluded E1 form. These results indicate that a cation permeable pathway, activated by inside positive potentials, can be ascribed to the Na-K pump--possibly through interaction with its gating mechanism.     

Evolution of the sodium pump

Eukaryotic plasma membranes (PMs) are energized by electrogenic P-type ATPases that generate either Na⁺ or H⁺ motive forces to drive Na⁺ and H⁺ dependent transport processes, respectively. For this purpose, animal rely on Na⁺/K⁺-ATPases whereas fungi and plants employ PM H⁺-ATPases. Prokaryotes, on the other hand, depend on H⁺ or Na⁺-motive electron transport complexes to energize their cell membranes. This raises the question as to why and when electrogenic Na⁺ and H⁺ pumps evolved? Here it is shown that prokaryotic Na⁺/K⁺-ATPases have near perfect conservation of binding sites involved in coordination of three Na⁺ and two K⁺ ions. Such pumps are rare in Eubacteria but are common in methanogenic Archaea where they often are found together with P-type putative PM H⁺-ATPases. With some exceptions, Na⁺/K⁺-ATPases and PM H⁺-ATPases are found everywhere in the eukaryotic tree of life, but never together in animals, fungi and land plants. It is hypothesized that Na⁺/K⁺-ATPases and PM H⁺-ATPases evolved in methanogenic Archaea to support the bioenergetics of these ancestral organisms, which can utilize both H⁺ and Na⁺ as energy currencies. Both pumps must have been simultaneously present in the first eukaryotic cell, but during diversification of the major eukaryotic kingdoms, and at the time animals diverged from fungi, animals kept Na⁺/K⁺-ATPases but lost PM H⁺-ATPases. At the same evolutionary branch point, fungi did loose Na⁺/K⁺-ATPases, and their role was taken over by PM H⁺-ATPases. An independent but similar scenery emerged during terrestrialization of plants: they lost Na⁺/K⁺-ATPases but kept PM H⁺-ATPases.     

Electrical and biochemical properties of an enzyme model of the sodium pump

The electrochemical properties of a widely accepted six-step reaction scheme for the Na+, K+-ATPase have been studied by computer simulation. Rate coefficients were chosen to fit the nonvectorial biochemical data for the isolated enzyme and a current-voltage (I-V) relation consistent with physiological observations was obtained with voltage dependence restricted to one (but not both) of the two translocational steps. The vectorial properties resulting from these choices were consistent with physiological activation of the electrogenic sodium pump by intracellular and extracellular sodium (Na+) and potassium (K+) ions. The model exhibited K+/K+ exchange but little Na+/Na+ exchange unless the energy available from the splitting of adenosine triphosphate (ATP) was reduced, mimicking the behavior seen in squid giant axon. The vectorial ionic activation curves were voltage dependent, resulting in large shifts in apparent Km's with depolarization. At potentials more negative than the equilibrium or reversal potential transport was greatly diminished unless the free energy of ATP splitting was reduced. While the pump reversal potential is at least 100 mV hyperpolarized relative to the resting potential of most cells, the voltage-dependent distribution of intermediate forms of the enzyme allows the possibility of considerable slope conductance of the pump I-V relation in the physiological range of membrane potentials. Some of the vectorial properties of an electrogenic sodium pump appear to be inescapable consequences of the nonvectorial properties of the isolated enzyme. Future application of this approach should allow rigorous quantitative testing of interpretative ideas concerning the mechanism and stoichiometry of the sodium pump.     

A two-site model for sodium transport in human erythrocytes

A kinetic model has been proposed for human erythrocytes to account for the responses of sodium transport to alterations in the extracellular sodium concentration in the presence and absence of potassium. The proposed model characterizes the movement of sodium from the outer surface of the erythrocyte membrane to the inner surface in terms of a carrier which has two sites with differing affinities for sodium ions.     

Sulfatide role in the sodium pump

Summary Sodium efflux was studied in²²Na-loaded red blood cells in the presence of arylsulfatase, an enzyme that specifically hydrolyzes sulfatide. Sodium efflux was inhibited in proportion to the amount of arylsulfatase present. Maximum inhibition was almost as high as the efflux obtained in medium with K⁺ absent. At maximum inhibition 83.2% of the sulfatide content of the fragmented red blood cell membranes was hydrolyzed and ouabain-sensitive (Na⁺+K⁺)-ATPase activity was inhibited by 100%. Sodium efflux, sulfatide content, and (Na⁺+K⁺)-ATPase activity were unaffected with arylsulfatase in the presence of a high concentration of sulfatide. These results indicate that sulfatide plays a specific role in sodium and potassium ion transport. They also suggest that most sulfatide is localized externally in the red blood cell membrane.     

The sodium pump. Its molecular properties and mechanics of ion transport.

The sodium pump (Na(+)/K(+)-ATPase; sodium- and potassium-activated adenosine 5'-triphosphatase; EC 3.6.1.37) has been under investigation for more than four decades. During this time, the knowledge about the structure and properties of the enzyme has increased to such an extent that specialized groups have formed within this field that focus on specific aspects of the active ion transport catalyzed by this enzyme. Taking this into account, this review, while somewhat speculative, is an attempt to summarize the information regarding the enzymology of the sodium pump with the hope of providing to interested readers from outside the field a concentrated overview and to readers from related fields a guide in their search for gathering specific information concerning the structure, function, and enzymology of this enzyme.     

Monensin A acid complexes as a model of electrogenic transport of sodium cation

New Monensin A acid complexes with water molecule, sodium chloride and sodium perchlorate were obtained and studied by X-ray and (1)H, (13)C NMR and FT-IR methods as well as ab initio calculations. The crystal structure of the complexes indicates the complexation of the water molecule and Na(+) cation in the pseudo-cycle conformation of the Monensin acid molecule stabilised by intramolecular hydrogen bonds. Important for stabilisation of this structure is also the intermolecular hydrogen bonds with water molecule or the coordination bonds with Na(+) cation. It is demonstrated that the counterions forming intermolecular hydrogen bonds with OH groups influence the strength of the intramolecular hydrogen bonds, but they have no influence on the formation of pseudo-cyclic structure. Spectroscopic studies of the complexes in dichloromethane solution have shown that the pseudo-cyclic structure of the compounds is conserved. As follows from the ab initio calculations, the interactions between the Na(+) cation and the electronegative oxygen atoms of Monensin acid totally change the molecular electrostatic potential around the supramolecular Monensin acid-Na(+) cationic complex relative to that of the neutral Monensin acid molecule.