Na(+)-dependent, active and Na(+)-independent, facilitated transport of formycin B in mouse spleen lymphocytes

Na(+)-dependent, active and Na(+)-independent facilitated nucleoside transport were characterized in mouse spleen cells using rapid kinetic techniques and formycin B, a metabolically inert analog of inosine, as substrate. The Michaelis-Menten constants for formycin B transport by the two transporters were about 30 and 400 microM, respectively. The first-order rate constant for Na(+)-dependent transport was about 4-times higher than that for facilitated formycin B transport. The Na(+)-dependent carrier is specific for uridine and purine nucleosides and accumulates formycin B concentratively in an unmodified form. Concentrative accumulation was inhibited by ATP depletion and gramicidin and ouabain treatment of the cells. Our data indicate a single Na(+)-binding site on the Na(+)-dependent nucleoside carrier and a Michaelis-Menten constant for Na+ of about 10 mM. This transporter was not significantly inhibited by dipyridamole and nitrobenzylthioinosine, inhibitors of the facilitated transporter. The Na(+)-independent, facilitated nucleoside transporter of spleen cells exhibits properties comparable to those of the carriers present in mammalian cells in general. The B lymphocytes remaining after depletion of spleen cell populations of T lymphocytes by incubation with a combination of T-cell specific monoclonal antibodies plus complement exhibited about the same activities of active and facilitated nucleoside transport as the original suspension.     

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.     

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high- affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta- gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.     

Active potassium transport coupled to active sodium transport in vesicles reconstituted from purified sodium and potassium ion-activated adenosine triphosphatase from the rectal gland of Squalus acanthias.

Vesicles containing a purified shark rectal gland (sodium + potassium)-activated adenosine triphosphatase-(NaK ATPase) were prepared by dialyzing for 2 days egg lecithin, cholate, and the NaK ATPase purified from the rectal gland of Squalus acanthias. These vesicles were capable of both Na+ and K+ transport. Studies of K+ transport were made by measuring the ATP-stimulated transport outward of 42K+ or 86Rb+. Vesicles were preloaded with isotope by equilibration at 4 degrees for 1 to 3 days. Transport of 42K+ or 86Rb+ was initiated by addition of MgATP to the vesicles. The ATP-dependent exit of either isotope was the same. Experiments are presented which show that this loss of isotope was not due to changes in ion binding but rather due to a loss in the amount of ion trapped in the vesicular volume. The transport of K+ was dependent on external Mg2+. CTP was almost as effective as ATP in stimulating K+ transport, while UTP was relatively ineffective. These effects of nucleotides parallel their effects on Na+ accumulation and their effectiveness as substrates for the enzyme. Potassium transport was inhibited by ouabain and required the presence of Na+. The following asymmetries were seen: (a) addition of external Mg2+ supported K+ transport; (b) ouabain inhibited K+ transport only if it was present inside the vesicles; (c) addition of external Na+ to the vesicles stimulated K+ transport. External Li+ was ineffective as a Na+ substitute. The specific requirement of external Na+ for K+ transport indicates that K+ exit is coupled to Na+ entry. Changes in the internal vesicular ion concentrations were studied with vesicles prepared in 20 mM NaCl and 50 mM KCl. After 1 hour of transport at 25 degrees, a typical Na+ concentration in the vesicles in the presence of ATP was 72 mM. A typical K+ concentration in the vesicles was 10 mM as measured with 42K+ or 6 mM as measured with 86Rb+. The following relationships have been calculated for Na+ transport, K+ transport and ATP hydrolysis: Na+/ATP = 1.42, K+/ATP =1.04, and Na+/K+ = 1.43. The ratio of 2.8 Na+ transported in to 2 K+ transported out is very close to the value reported for the red cell membrane. Potassium-potassium exchange similar to that observed in the red cell membrane and attributed to the Na+-K+ pump (stimulated by ATP and orthophosphate and inhibited by ouabain) was observed when vesicles were prepared in the absence of Na+. The results reported in this paper prove that the shark rectal gland NaK ATPase, which is 90 to 95% pure, is the isolated pump for the coupled transports of Na+ and K+.     

Proton diet for the sodium pump

In the absence of Na(+) and K(+) ions the Na,K-ATPase shows a pH-dependent ATP hydrolysis that can be inhibited by ouabain. At pH 7.2 this activity is 5% of the maximal under physiological conditions. It could be inferred that this activity is associated with H(+) transport in both directions across the membrane and facilitates an H-only mode of the sodium pump under such unphysiological conditions. By the analysis of experiments with reconstituted proteoliposomes an overall electroneutral transport mode has been proven. The stoichiometry was determined to be 2 H(+)/2 H(+)/1 ATP and is comparable to what is known from the closely related H,K-ATPase. By time-resolved ATP-concentration jump experiments it was found that at no time was the third, Na(+)-specific binding site of the pump occupied by protons. A modified Post-Albers pump cycle is proposed, with H(+) ions as congeners for Na(+) and K(+), by which all experiments performed can be explained.     

Autoregulation of the electrogenic sodium pump

The dependence of electrogenic sodium pump activity on changes in the cell volume of Helix pomatia neurons with different levels of intracellular sodium ion concentration was studied. Hypertonic solutions caused hyperpolarization of the membrane and increased membrane resistance in cells with a low sodium content (low-sodium cells; LSC). The activity of the electrogenic sodium pump in hypertonic solutions was increased compared to the activity in hypotonic solutions in LSC and decreased in cells with a high sodium content (high-sodium cells; HSC). The concentration of ouabain which led to maximal inhibition of active 22Na efflux from the neurons was 10(-4) M. Lower concentrations of ouabain (10(-8) M and lower) did not inhibit the sodium pump but stimulated it. The swelling of neurons in hypotonic solutions was accompanied by an increase in the number of binding sites for ouabain, while shrinking in hypertonic solutions led to the opposite effect--a decrease in binding sites. An increase in the number of binding sites also took place in normal isotonic potassium-free solutions compared with normal Ringer's solution. Two saturable components of ouabain binding were detectable in all solutions examined. gamma-Aminobutyric acid (GABA) and acetylcholine (ACh) increased the number of ouabain binding sites on the membrane. The results suggest that there are two opposite mechanisms by which cell volume changes can modulate the pump activity. One of them depends on the intracellular sodium ion concentration and causes pump activation in hypertonic solutions in LSC and saturation in HSC, while a second mechanism mediates the activating effect of cell swelling on the sodium pump in HSC. In addition, there may be a negative feedback between the pump activity and the number of functioning pump units in the membrane.     

Regulation of internal pH of sea urchin sperm. A role for the Na/K pump

In the absence of sodium, sea urchin sperm have an acidic internal pH. The addition of sodium, lithium, or ammonium, but not of potassium ions, induces an internal alkalization. If potassium is added in the presence of sodium, a further alkalization is obtained; in contrast, potassium addition in presence of Li+ or NH+4 does not change the internal pH. The K+-induced pHi change is inhibited by ouabain and when sperm are depleted of their ATP. A large part of the potassium influx is stimulated by Na+, but not Li+, and inhibited by ouabain and cellular ATP depletion. We conclude that activity of Na/K-ATPase pumps located in the plasma membrane of sea urchin sperm could play a role in regulating the internal pH of sea urchin sperm by recycling sodium ions that enter the cell through Na/H countermovements.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Sodium transport in basolateral membrane vesicles from rat enterocytes

Basolateral membranes purified from rat jejunal enterocytes and enriched 14 times in (Na, K)-ATPase, are present as unsealed and right side out (RSO) or inside out (IO) vesicles in the ratio 2:2:1, as determined by detergent activation of ATPase activity. Entrance of 1 mM Na into basolateral membrane vesicles was measured in the presence and in the absence of 5 mM ATP by a rapid filtration technique, under different experimental conditions. Carrier-mediated Na transport across the basolateral membrane can be trans-stimulated and cis-inhibited by K and further stimulated by ATP (activation of the Na pump). The ATP effect can be suppressed by vanadate and strophanthidin and enhanced by bleomycin (19% increase), which positively also acts on (Na, K)-ATPase activity (16% increase). In addition to the Na pump this study demonstrates the existence of a carrier-mediated Na transport trans-stimulated by K. There appears to be no cotransport of Na-K.     

Neutralization of the aspartic acid residue Asp-367, but not Asp-454, inhibits binding of Na+ to the glutamate-free form and cycling of the glutamate transporter EAAC1

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na(+) ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na(+) cotransport. To test the effect of charge neutralization mutations in these positions on Na(+) binding to the glutamate-free transporter, we recorded the Na(+)-induced anion leak current to determine the K(m) of EAAC1 for Na(+). For EAAC1(WT), this K(m) was determined as 120 mm. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na(+) activated the anion leak current with a K(m) of about 2 m, indicating dramatically impaired Na(+) binding to the mutant transporter. In contrast, the Na(+) affinity of EAAC1(D454N) was virtually unchanged compared with the wild type transporter (K(m) = 90 mm). The reduced occupancy of the Na(+) binding site of EAAC1(D367N) resulted in a dramatic reduction in glutamate affinity (K(m) = 3.6 mm, 140 mm [Na(+)]), which could be partially overcome by increasing extracellular [Na(+)]. In addition to impairing Na(+) binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na(+) in the glutamate-free transporter form.     

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.     

Glutamic acid 472 and lysine 480 of the sodium pump alpha 1 subunit are essential for activity. Their conservation in pyrophosphatases suggests their involvement in recognition of ATP phosphates.

P-type ATPases such as the Na+,K+-ATPase (sodium pump) hydrolyze ATP to pump ions through biological membranes against their electrochemical gradients. The mechanisms that couple ATP hydrolysis to the vectorial ion transport are not yet understood, but unveiling structures that participate in ATP binding and in the formation of the ionophore might help to gain insight into this process. Looking at the alpha- and beta-phosphates of ATP as a pyrophosphate molecule, we found that peptides highly conserved among all soluble inorganic pyrophosphatases are also present in ion-transporting ATPases. Included therein are Glu48 and Lys56 of the Saccharomyces cerevisiae pyrophosphatase (SCE1-PPase) that are essential for the activity of this enzyme and have been shown in crystallographic analysis to interact with phosphate molecules. To test the hypothesis that equivalent amino acids are also essential for the activity of ion-transporting ATPases, Glu472 and Lys480 of the sodium pump alpha 1 subunit corresponding to Glu48 and Lys56 of SCE1-PPase were mutated to various amino acids. Mutants of the sodium pump alpha1 subunit were expressed in yeast and analyzed for their ATPase activity and their ability to bind ouabain in the presence of either ATP, Mg2+, and Na+ or phosphate and Mg2+. All four mutants investigated, Glu472Ala, Glu472Asp, Lys480Ala, and Lys480Arg, display only a fraction of the ATPase activity obtained with the wild-type enzyme. The same applies with respect to their ability to bind ouabain, where maximum ouabain binding to the mutants accounts for only about 10% of the binding obtained with the wild-type enzyme. On the basis of our results, we conclude that Glu472 and Lys480 are essential for the activity of the sodium pump. Their function is probably to arrest the alpha- and beta-phosphate groups of ATP in a proper position prior to hydrolysis of the gamma-phosphate group. The identification of these amino acids as essential components of the ATP-recognizing mechanism of the pump has resulted in a testable hypothesis for the initial interactions of the sodium pump, and possibly of other P-type ATPases, with ATP.     

Mg2+-ATP-dependent sodium transport in inside-out basolateral plasma membrane vesicles from guinea-pig small intestinal epithelial cells

The transport of sodium into inside-out basolateral plasma membrane vesicles from small intestinal epithelial cells has been examined. It was found, under equilibrium conditions, that binding of 22Na represents approx. 55% of the total uptake during an equilibration period of 30 min; 45% of the total uptake correspond to passive sodium entry in the vesicle space. In addition to binding and to passive Na+ entry, two distinct mechanisms capable of accumulating sodium in the intravesicular space can be demonstrated when ATP is added to the incubation medium. One transports sodium actively in the absence of potassium, whereas the other requires the presence of potassium in the interior of the vesicles. The two mechanisms can also be differentiated by their affinities for sodium, their optimal pH and by their behaviour towards different inhibitors. Thus, the mechanism that transports sodium in the absence of potassium is refractory to ouabain, but is inhibited by ethacrynic acid and furosemide, whilst the mechanism that accumulates sodium inside the vesicles in the presence of internal potassium is strongly inhibited by ouabain, is weakly inhibited by ethacrynic acid and is insensitive of furosemide. ATP is a specific stimulator of both processes, and the requirement for magnesium is absolute in both cases.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

Regulation of neuronal bioenergy homeostasis by glutamate

Bioenergy homeostasis is crucial in maintaining normal cell function and survival and it is thus important to understand cellular mechanisms underlying its regulation. Neurons use a large amount of ATP to maintain membrane potential and synaptic communication, making the brain the most energy consuming organ in the body. Glutamate mediates a large majority of synaptic transmission which is responsible for the expression of neural plasticity and higher brain functions. Most of the energy cost is attributable to the glutamatergic system; under pathological conditions such as stroke and brain ischemia, neural energy depletion is accompanied by a massive release of glutamate. However, the specific cellular processes implicated in glutamate-dependent bioenergy dynamics are not well understood. We find that glutamate induces a rapid and dramatic reduction of ATP levels in neurons, through reduced ATP genesis and elevated consumption. ATP reduction depends on NMDA receptor activity, but is not a result of neuronal firing, gap junction-mediated leaking or intracellular signaling. Similar changes in ATP levels are also induced by synaptic glutamate accumulation following suppression of glutamate transporter activity. Furthermore, the glutamate-induced ATP down-regulation is blocked by the sodium pump inhibitor ouabain, suggesting the sodium pump as the primary energy consumer during glutamate stimulation. These data suggest the important role of glutamate in the control of cellular ATP homeostasis.     

Direct measurement of the membrane potential of Ehrlich ascites tumor cells: lack of effect of valinomycin and ouabain.

The membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control cells was 12.0 mV, inside negative. Neither valinomycin nor ouabain alone affected this value. However, valinomycin and ouabain in combination resulted in a slight hyperpolarization of the membrane. Concomitant determinations of cellular Na+, K+ and Cl- showed that valinomycin induced net losses of K+ and Cl- and a net gain in Na+ when compared to ouabain-inhibited cells. K+ permeability was increased by approximately 30% in the presence of valinomycin. In addition, valinomycin caused a rapid depletion of cellular ATP. Inhibition of Na/K transport by ouabain was without sparing effect on the rate of ATP depletion. Possible mechanisms for the electroneutral increase in K+ permeability induced by valinomycin are discussed.     

Effects of aldosterone on (Na+ + K+)-ATPase of amphibian sodium-transporting epithelial cells (A6) in culture

Stimulation by aldosterone of sodium reabsorption can be reproduced on a cell line, A6, derived from the renal tissue of Xenopus laevis. These cells organize themselves as a polarized epithelium carrying out unidirectional sodium transport, reflected by the short-circuit current (Isc). Isc response to aldosterone starts to be apparent after a latency period of 2-3 h; the full hormonal effect takes much longer. On the other hand, (Na+ + K+)-ATPase activity and density in ouabain binding sites did not increase before several hours of treatment. At that stage, while Isc more than trebled, Na+ pump activity and density went up by less than 50%. A significant influence of aldosterone on the way the Na+ pump operates is considered unlikely, since cell interaction with ouabain remained unchanged (Kd approximately 18 nM). Furthermore, the close correspondence of hormonal effect, in relative terms, on (Na+ + K+)-ATPase activity vs density, argues against a significant degree of recruitment of spare pump units. Thus aldosterone effect on Na+ pump probably results from increased biosynthesis of the enzyme. The aldosterone dependent Na+ pump stimulation is apparently unrelated to sodium available for transport. The hormone seems to act on Na+ pump directly.     

A proton-mediated conformational shift identifies a mobile pore-lining cysteine residue (Cys-561) in human concentrative nucleoside transporter 3

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na(+)-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H(+)-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H(+), but not Na(+), and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H(+)-induced conformational state of the protein.     

Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches.

Strophanthidin inhibits KATP channels in 2,4-dinitrophenol-poisoned heart cells (). The current study shows that the Na/K pump interacts with KATP current (IK-ATP) via submembrane ATP depletion in isolated giant membrane patches and in nonpoisoned guinea pig cardiac cells in whole-cell configuration. IK-ATP was inhibited by ATP, glibenclamide, or intracellular Cs+. Na/K pump inactivation by substitution of cytoplasmic Na+ for Li+ or N-methylglucamine decreased both IK-ATP by 1/3 (1 mM ATP, zero calcium), and IC50 of ATP for IK-ATP (0.3 +/- 0.1 mM) by 2/5. The Na+/Li+ replacement had no effect on IK-ATP at low pump activity ([ATP] </= 0.1 mM or 100 microM ouabain) or when IK-ATP was completely inhibited by 10 mM ATP. In whole-cell configuration, ouabain inhibited up to 60% of inwardly rectifying IK-ATP at 1 mM ATP in the pipette but not at 10 mM ATP and 10 mM phosphocreatine when IK-ATP was always blocked. However, mathematical simulation of giant-patch experiments revealed that only 20% of ATP depletion may be attributed to the ATP concentration gradient in the bulk solution, and the remaining 80% probably occurs in the submembrane space.     

Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.