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.     

An electrostatic model of a membrane ion pump

The model is based upon an ion channel with an electric dipolar structure. With simplifying assumptions it is possible to calculate that a typical channel, 1 nm in diameter and 5 nm long, could contain at most two or three univalent cations at a time. The channel ion binding sites have an effective affinity for ions from the fluid bathing the negative end of the channel, several orders of magnitude higher than their affinity for ions from the fluid bathing the positive end of the channel. The approach of an external, positively charged body to the negative end of the channel, is sufficient to convert the two- or three-channel ion sites with high affinity for ions from the fluid bathing this end into very low affinity sites for the same ions that now have access only to the fluid bathing the other end of the channel. The change in affinity and fluid access requires no molecular or electrical change in the channel structure other than the passive superposition of the electrostatic potential of the dipolar channel and that of the charged body. An oscillating electric field externally applied to an electric dipolar channel is shown to result in the unidirectional pumping of cations in the direction of the channel dipole even against large adverse ion concentration gradients. The energy required must be supplied by the sources of the electric field. By using two such channels in close proximity, one selective for K+ ions with its dipole moment pointing into a cell and the other selective for Na+ ions with its dipole moment pointing out from the cell, it is possible to construct a model pump with calculated properties that simulate many of those measured for Na+-K+-ATPase, with both physiological and artificial ionic concentrations.     

A cytochrome that can pump sodium ion

Previous studies have shown that the bacterium, Vitreoscilla, generates a respiratory-driven delta psi Na+. Two major respiratory electron transport proteins, NADH dehydrogenase (NADH:Quinone oxidoreductase), and cytochrome o terminal oxidase are candidates for the electrogenic Na+ pumping that mediates the delta psi Na+ formation. The NADH oxidase activity of the membranes was enhanced more by Na+ than by Li+. The NADH:Quinone oxidoreductase activity in the respiratory chain was enhanced by Na+ and Li+, whereas the quinol oxidase activity of cytochrome o was enhanced specifically by Na+, and not by Li+, K+, or choline. Purified cytochrome o, reconstituted into Na(+)-loaded liposomes in the right-side-out orientation, catalyzed a net Na+ extrusion when energized with Q1H2(1). In nonloaded inside-out proteoliposomes, this cytochrome catalyzed a net uptake of 22Na+ when energized with ascorbate/TMPD. Both Na(+)-pumping activities were inhibited by CN-. These results are consistent with the Vitreoscilla cytochrome o being a redox-driven Na+ pump.     

A physical analysis of the ion parametric resonance model

We show, in elementary terms, using for the most part only elementary mathematics, the physical bases for the ion parametric resonance model so as to clarify the assumptions and consequences of the model. The analysis shows why, contrary to earlier conclusions, no combination of weak DC and AC magnetic fields can modify the transition rate to the ground state of excited ions. Although reinterpretations of the biological consequences of the motion of the excited ions circumvent that particular objection to the model, those changes introduce other difficulties. Also, other objections to the mechanism still stand; hence the model cannot account for any purported biological effects of weak extremely low frequency magnetic fields. Bioelectromagnetics 19:181–191, 1998. © 1998 Wiley-Liss, Inc.     

A rotary nano ion pump: a molecular dynamics study

The dynamics of a rotary nano ion pump, inspired by the F (0) part of the F(0)F(1)-ATP synthase biomolecular motor, were investigated. This nanopump is composed of a rotor, which is constructed of two carbon nanotubes with benzene rings, and a stator, which is made of six graphene sheets. The molecular dynamics (MD) method was used to simulate the dynamics of the ion nanopump. When the rotor of the nanopump rotates mechanically, an ion gradient will be generated between the two sides of the nanopump. It is shown that the ion gradient generated by the nanopump is dependant on parameters such as the rotary frequency of the rotor, temperature and the amounts and locations of the positive and negative charges of the stator part of the nanopump. Also, an electrical potential difference is generated between the two sides of the pump as a result of its operation.     

Evaluation of whole-animal data using the ion parametric resonance model

Changes observed in the behavioral response of land snails from exposure to parallel ac and dc magnetic fields demonstrate limited agreement with the predictions of an interaction model proposed by Lednev and the predictions of a recently proposed ion parametric resonance (IPR) model. However, the inadequate number of reported data points, particularly in a critical exposure range, prevents unambiguous application of either the Lednev or the IPR model. © 1995 Wiley-Liss, Inc.     

Electrogenesis in a model of "boundary transport" in the sodium pump]

An equation of the membrane potential (MP) was developed for a model of sodium pump when it was assumed that a macromolecule-carrier operated on the interior boundary of the membrane exchaning Na+i for K+ of membrane. These ions then moved downhill the electrochemical gradient inside the membrane. There are factors in the equation which indicate energetical consumtions for the ionic exchange. In was shown that an exchange of 3Na+ for 2K+ required 9,0 kkal/mole. The equation satisfactorily describes experimental values of MP during the electrogenic active transport. After all it was shown that an alteration of MP for 100 mV did not affect the operation of sodium pump i.e. the influence of outer electrical field was not detected.     

Sequence of the sodium ion pump oxaloacetate decarboxylase from Salmonella typhimurium.

A genomic library of Salmonella typhimurium DNA was constructed in the lambda-phage EMBL3 and screened by immunoblotting for expression of the oxaloacetate decarboxylase alpha-subunit. After subcloning on plasmids the entire sequence of the oxaloacetate decarboxylase was determined. The genes encoding subunits gamma (oadG), alpha (oadA), and beta (oadB) of the decarboxylase are clustered on the chromosome in that order. A typical consensus sequence of a promoter is not found upstream of the oadG gene, but putative ribosome binding regions can be identified before each subunit gene. The amino acid sequences are highly homologous to those of oxaloacetate decarboxylase from Klebsiella pneumoniae with 71% identity between the gamma-subunits, 92% identity between the alpha-subunits, and 93% identity between the beta-subunits. The homology between the corresponding beta-subunits appeared to exist only between the 312 N-terminal amino acid residues. It was shown that a cloning artifact has occurred during DNA sequence determination of the beta-subunit from K. pneumoniae and has led to erroneous results. The sequence of this polypeptide is corrected in the Appendix to this paper. A plasmid encoding the three oad genes and that for the anaerobic citrate carrier (citS) was cloned from the chromosomal DNA and used for sequence determination.     

Isolation and characterization of oxaloacetate decarboxylase of Salmonella typhimurium,a sodium ion pump

Anaerobic growth of Salmonella typhimurium on citrate is Na⁺-dependent and requires induction of the necessary enzymes during a 20–40 h lag phase. The citrate fermentation pathway involves citrate lyase and oxaloacetate decarboxylase. The decarboxylase is a membrane-bound. Na⁺-activated, biotin-containing enzyme that functions as a Na⁺ pump. Oxaloacetate decarboxylase was isolated by affinity chromatography of a Triton X-100 extract of the bacterial membranes on avidin-Sepharose. The enzyme consists of three subunits , , , with apparent molecular weights of 63800, 34500 and 10600. The -chain contains a covalently attached biotin group and binds to antibodies raised against the -subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae. The Na⁺ transport function was reconstituted by incorporation of the puriried enzyme into proteoliposomes.     

Autoradiographic localization of tritiated ouabain-sensitive sodium pump sites in ion transporting epithelia

The freeze-dry autoradiographic method devised originally by Stirling (J Cell Biol 53:704, 1972) to localize Na+ pump sites with (3H)ouabain is reviewed. Biochemical, physiological, and autoradiographic data are discussed which establish that ouabain binding to intact tissue conforms to rigid criteria for high Na+ pump specificity. Among these are that glycoside binding exhibits saturation kinetics, ligand dependence, and close correlation with degrees of inhibition of Na+-K+-ATPase and Na+ transport. Moreover, localization of Na+ pump sites by this technique shows a cell and membrane specificity which mirrors that obtained by cytochemical and immunocytochemical methods. In addition to resolving cell-specific patterns of localization in heterogeneous tissues, the demonstration of Na+-K+-ATPase by these techniques indicates that Na+ pumps are distributed uniformly along plasmalemmal surfaces and are restricted to the basolateral interface in reabsorptive and secretory epithelia despite the opposing polarity of net transepithelial electrolyte transport.     

Crystal structure of the carboxyltransferase subunit of the bacterial sodium ion pump glutaconyl-coenzyme A decarboxylase

Glutaconyl-CoA decarboxylase is a biotin-dependent ion pump whereby the free energy of the glutaconyl-CoA decarboxylation to crotonyl-CoA drives the electrogenic transport of sodium ions from the cytoplasm into the periplasm. Here we present the crystal structure of the decarboxylase subunit (Gcdalpha) from Acidaminococcus fermentans and its complex with glutaconyl-CoA. The active sites of the dimeric Gcdalpha lie at the two interfaces between the mono mers, whereas the N-terminal domain provides the glutaconyl-CoA-binding site and the C-terminal domain binds the biotinyllysine moiety. The Gcdalpha catalyses the transfer of carbon dioxide from glutaconyl-CoA to a biotin carrier (Gcdgamma) that subsequently is decarboxylated by the carboxybiotin decarboxylation site within the actual Na(+) pump (Gcdbeta). The analysis of the active site lead to a novel mechanism for the biotin-dependent carboxy transfer whereby biotin acts as general acid. Furthermore, we propose a holoenzyme assembly in which the water-filled central channel of the Gcdalpha dimer lies co-axial with the ion channel (Gcdbeta). The central channel is blocked by arginines against passage of sodium ions which might enter the central channel through two side channels.     

The parametric pump mechanism in separation of components in heterogeneous systems. I. Macroscopic distributed systems.

A dynamic method is proposed for the separation of the electrolyte components using a parametric pump with an ion exchange column. It was studied experimentally and described mathematically. The parametric separation of mixtures is based on interactions of two oscillating fields with a heterogeneous system containing two phases, a liquid and a solid one, the components of the mixture being able to redistribute between the phases. The field of mechanical force is responsible for cyclic relative displacement of the phases, and synchronously changing temperature causes redistribution of the components between them. This results in sodium and potassium fluxes opposite in direction which in turn leads to accumulation of sodium and potassium in opposite end cells.     

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.