Effects of cytoplasmic sodium concentration on the electrogenicity of the sodium pump

Inside-out membrane vesicles were prepared from human red blood cells pretreated with diisothiocyano-2,2'-disulfonic stilbene to inhibit anion fluxes. The pH-sensitive probe fluorescein isothiocyanate-dextran was incorporated inside the vesicles. Formation of pH gradients due to proton transport by the sodium pump was distinguished from pH gradients formed in response to transmembrane electrical potentials generated by the pump by virtue of their insensitivity and sensitivity, respectively, to dissipation by lipophilic cations. Under the conditions used (pH 6.6), proton transport by the Na,K-ATPase was minimized, and the formation of pH gradients in response to electrical potentials was detected. Thus, the generation of a strophanthidin-sensitive, ATP-dependent electrical potential, inside positive (approximately 1 mV) upon addition of 4 meq of sodium to potassium-filled inside-out vesicles is consistent with the well documented stoichiometry of three sodium ions exchanging with two potassium ions. In contrast, when the cytoplasmic sodium concentration is reduced to less than or equal to 0.4 mM, the potential generated is of the opposite sign, i.e. inside negative, consistent with the decreased Na:K coupling ratio reported previously, i.e. Na:K(Rb) coupling ratios of approximating 1:2 when the sodium concentration is reduced to 0.2 mM (Blostein, R. (1983) J. Biol. Chem. 258, 12228-12232).     

The influence of external sodium ions on the sodium pump in erythrocytes

1. A study has been made of the interaction between Na(+) and K(+) on the adenosine triphosphatase activity of erythrocyte ;ghosts', and on the K(+) influx and Na(+) efflux of intact erythrocytes. The adenosine triphosphatase activity and the ion movements were greater at a low external K(+) concentration in the absence of Na(+) than they were in the presence of 150mm-Na(+). The inhibition by external Na(+) of K(+) influx had an inhibitory constant of 5-10mm. 2. Activation by K(+) of kidney microsomal adenosine triphosphatase was retarded by Na(+), and activation by Na(+) was retarded by K(+). Fragmented erythrocyte membranes behaved similarly. 3. These observations suggest that there is competition between Na(+) and K(+) at the K(+)-sensitive site of the membrane.     

Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum

Coupling of Ca2+ transport to ATP hydrolysis by isolated skeletal muscle sarcoplasmic reticulum vesicles has been investigated by means of ATP pulse methods. The stoichiometric amounts of Ca2+ transported per pulse of ATP were measured by Ca2+-stat methods, using either a Ca2+ electrode or arsenazo III as end point detectors, or by means of 45CaCl2. Maximum coupling ratios (Ca2+/ATP), of 1.82 +/- 0.13 occurred at pH 6.8, 25 degrees C, and in the presence of saturating Ca2+ concentrations. Ca2+/ATP values decreased at alkaline pH, with an apparent pK alpha of 7.9. The coupling ratio was unaltered between 6 and 30 degrees C, but decreased to 0.4 at 42 degrees C. Uncoupling by alkaline pH and high temperatures was reversible. The coupling process was Ca2+-dependent, with a K0.5 value for Ca2+ of 0.12 microM and a Hill coefficient of 2.0. Ca2+ ions, which were transported into vesicles under conditions resulting in low coupling ratios, were retained as the calcium oxalate precipitate, following complete hydrolysis of substrate. Passive Ca2+ efflux and Ca2+ exchange, were independent of pH. The observed variations in Ca2+/ATP ratio cannot readily be explained on the basis of a pump-leak model. Rather, the Ca2+-ATPase appears to be capable of pumping Ca2+ ions, under physiological conditions, with variable stoichiometry that is dependent upon its thermodynamic loading.     

Hydrogen-bonding network at the cytoplasmic region of a light-driven sodium pump rhodopsin KR2

Light-driven sodium-pumping rhodopsins are able to actively transport sodium ions. Structure/function studies of Krokinobacter eikastus rhodopsin 2 (KR2) identified N61 and G263 at the cytoplasmic surface constituting the “Ion-selectivity filter” for sodium ions, while retinal Schiff base acts as the light “Switch and Gate” for transport of sodium ions. Q123 is located between the two regions, and plays an important role for the pump function, which was implicated by functional, spectroscopic, X-ray crystallographic and computational studies. According to the atomic structure of KR2, Q123 is involved in the hydrogen-bonding network at the cytoplasmic region, together with S64, protein-bound waters, and peptide carbonyl of K255 bound to the chromophore. To gain the detailed structural information around Q123, here we compared light-induced difference Fourier-transform infrared (FTIR) spectra at 77?K between the wild-type (WT) and mutant proteins of KR2, such as Q123A, Q123V, and S64A. The obtained spectra were very similar between WT and these mutants, whereas the observed mutation effects enabled us to identify vibrations of the hydrogen-bonding network at the Q123 and S64 region. This is unique for KR2, not for the corresponding mutations in a light-driven proton-pump bacteriorhodopsin (BR). Hydrogen-bonding alteration is absent for the mutants of KR2, suggesting that proper inter-helical connectivity of helices B, C, and G is important for protein structural changes for sodium-pump function, which is controlled by the region around Q123.     

Quantitative measurement of the influence of plasma sodium concentration on the overall tubular reabsorption of sodium.

Comparative experiments on isolated dog kidneys perfused with heparinized blood with or without dilution of the blood by isotonic or hypotonic saline demonstrated that the fractional excretion of sodium is modulated positively by plasma sodium concentration. This relationship was evaluated quantitatively and corresponded to the values found in the whole animal. The renal response to the variations of plasma sodium concentration is therefore autonomous and its mediation by extrarenal natriuretic or antinatriuretic factors cannot be demonstrated in the narcotized animal.     

The other functions of the sodium pump

Na/K-ATPase (the sodium pump) was discovered in the 1950s as the plasma membrane enzyme that carries out the coupled active transports of Na⁺ and K⁺ across the membranes of nearly all eukaryotic cells. It was not until the 1990s when it was shown that besides pumping ions, Na/K-ATPase is also capable of stimulus-induced interactions with neighboring proteins that lead to activations of signal transduction pathways causing cell growth. This article is an attempt to review the progress of the research on these signaling functions of sodium pump during the past 2–3 decades. The covered topics include (a) the controversial digitalis-induced growth activations through the epidermal growth factor receptor and Src kinase in cardiac myocytes and several other cell types; (b) the extensive findings on digitalis-induced growth activations in cardiac myocytes and other cell types through phosphatidylinositol 3-kinases; and (c) a number of interesting but insufficiently studied signaling functions of the sodium pump.     

Transmembrane effects in the sodium pump system. I. The effect of external potassium and rubidium on the dependence of sodium efflux on sodium concentration in the frog muscle

The dependence of sodium efflux on intracellular sodium content with various potassium and rubidium concentration in the external medium has been studied on frog sartorious muscle. In potassium-sodium-free magnesium medium ouabain-sensitive sodium efflux was shown to be proportional to internal sodium concentration. In the presence of external ribidium (0.5--5.0 mM) the efflux concentration relations are non-linear, being closely described by assuming that 3 Na+ are transported per pump cycle. In sodium loaded muscles the efflux concentration curve was found to be dependent on the external rubidium concentration, becoming linear instead of S-shaped with the decrease in internal rubidium concentration from 5.0--2.5 to 1.0--0.5 mM. The apparent affinity constant for the internal sodium pump site increased with increasing the external rubidium (potassium) concentration. The data obtained may contribute to the kinetic evaluation of the type of Na-K pump mechanism, being more consistent with simultaneous model of pump operation.     

Transmembrane effects in the sodium pump system. II. The ratio of the sodium efflux to the sodium concentration in frog muscle in media with various sodium substitutes

The dependence of sodium efflux on the internal sodium concentration on sodium-free magnesium, Tris, coline and lithium media was investigated on frog striated muscle. In all the sodium-substituted media, the efflux concentration curve was found to be dependent on the external rubidium concentration, being S-shaped at the saturating external rubidium (potassium) concentration and becoming close to linear at the low external rubidium concentration (0.5-1.0 microM). The maximal sodium efflux at saturating levels of internal sodium concentrations remains unchanged with various sodium substitutes in the medium, whereas the affinity constant of internal sodium sites is dependent on the external cations.     

The proton pump of cytochrome c oxidase and its stoichiometry.

The operation of cytochrome c oxidase with ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine as substrate in antimycin-A-inhibited rat liver mitochondria is coupled to proton ejection. Measurements of the initial rate of valinomycin-dependent K+ uptake have shown that nearly 4 K+ are taken up as 2 electrons are transferred from cytochrome c to oxygen. This proves directly that a charge separation of nearly 4 occurs across the inner mitochondrial membrane each time 2 electrons are transferred to oxygen. Measurements of the initial rate of proton movement after addition of the reductant show that about 1.6 protons are released by the mitochondria as 2 electrons are transferred from cytochrome c to oxygen. The data support the suggestion of a proton pump coupled to the operation of cytochrome c oxidase [Wikstr?m, M. F. K. (1977) Nature (Lond.) 266, 271--273].