Sodium leak pathway and substrate binding order in the Na+-glucose cotransporter.

The Na+-glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes was shown to generate a phlorizin-sensitive sodium leak in the absence of sugars. Using the current model for SGLT1, where the sodium leak was presumed to occur after two sodium ions are bound to the free carrier before glucose binding, a characteristic concentration constant (Kc) was introduced to describe the relative importance of the sodium leak versus Na+-glucose cotransport currents. Kc represents the glucose concentration at which the Na+-glucose cotransport current is equal to the sodium leak. As both the sodium leak and the Na+-glucose cotransport current are predicted to occur after the binding of two sodium ions, the model predicted that Kc should be sodium-independent. However, by using a two-microelectrode voltage-clamp technique, the observed Kc was shown to depend strongly on the external sodium concentration ([Na+]o): it was four times higher at 5 mM [Na+]o than at 20 mM [Na+]o. In addition, the magnitude of the sodium leak varied as a function of [Na+]o in a Michaelian fashion, and the sodium affinity constant for the sodium leak was 2-4 times lower than that for cotransport in the presence of low external glucose concentrations (50 or 100 microM), whereas the current model predicted a sigmoidal sodium dependence of the sodium leak and identical sodium affinities for the sodium leak and the Na+-glucose cotransport. These observations indicate that the sodium leak occurs after one sodium ion is associated with the carrier and agree with predictions from a model with the binding order sodium-glucose-sodium. This conclusion was also supported by experiments performed where protons replaced Na+ as a "driving cation."     

Stoichiometry and voltage dependence of the sodium pump in voltage- clamped, internally dialyzed squid giant axon

The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG-sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K-free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct regulation of Na(+)-dependent myo-inositol transport by sugars in retinal pigment epithelium: role of phorbol ester and staurosporin

An Na(+)-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, alpha-methyl-D-glucoside (alpha MG); 1-5 mM nonradioactive MI, pyruvate, or lactate; or 0.2-20 microM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate 3H-MI (ratios of intracellular transported radioactive MI, [MI]i, to external free MI concentration, [MI]i/[MI]o) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM alpha MG, respectively, compared to cells grown in basic growth media. The rate of uptake of 3H-MI also was reduced to 63 +/- 15% or 48 +/- 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [3H]phlorizin was reduced to 52 +/- 7%, 61 +/- 5%, or 38 +/- 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM alpha MG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 +/- 8% reduction in [3H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12-14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12-14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [3H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes

Na/K pump current was determined between -140 and +60 mV as steady-state, strophanthidin-sensitive, whole-cell current in guinea pig ventricular myocytes, voltage-clamped and internally dialyzed via wide-tipped pipettes. Solutions were designed to minimize all other components of membrane current. A device for exchanging the solution inside the pipette permitted investigation of Na/K pump current-voltage (I-V) relationships at several levels of pipette [Na] [( Na]pip) in a single cell; the effects of changes in external [Na] [( Na]o) or external [K] [( K]o) were also studied. At 50 mM [Na]pip, 5.4 mM [K]o, and approximately 150 mM [Na]o, Na/K pump current was steeply voltage dependent at negative potentials but was approximately constant at positive potentials. Under those conditions, reduction of [Na]o enhanced pump current at negative potentials but had little effect at positive potentials: at zero [Na]o, pump current was only weakly voltage dependent. At 5.4 mM [K]o and approximately 150 mM [Na]o, reduction of [Na]pip from 50 mM scaled down the sigmoid pump I-V relationship and shifted it slightly to the right (toward more positive potentials). Pump current at 0 mV was activated by [Na]pip according to the Hill equation with best-fit K0.5 approximately equal to 11 mM and Hill coefficient nH approximately equal to 1.4. At zero [Na]o, reduction of [Na]pip seemed to simply scale down the relatively flat pump I-V relationship: Hill fit parameters for pump activation by [Na]pip at 0 mV were K0.5 approximately equal to 10 mM, nH approximately equal to 1.4. At 50 mM [Na]pip and high [Na]o, reduction of [K]o from 5.4 mM scaled down the sigmoid I-V relationship and shifted it slightly to the right: at 0 mV, K0.5 approximately equal to 1.5 mM and nH approximately equal to 1.0. At zero [Na]o, lowering [K]o simply scaled down the flat pump I-V relationships yielding, at 0 mV, K0.5 approximately equal to 0.2 mM, nH approximately equal to 1.1. The voltage-independent activation of Na/K pump current by both intracellular Na ions and extracellular K ions, at zero [Na]o, suggests that neither ion binds within the membrane field. Extracellular Na ions, however, seem to have both a voltage-dependent and a voltage-independent influence on the Na/K pump: they inhibit outward Na/K pump current in a strongly voltage-dependent fashion, with higher apparent affinity at more negative potentials (K0.5 approximately equal to 90 mM at -120 mV, and approximately 170 mM at -80 mV), and they compete with extracellular K ions in a seemingly voltage-independent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Access channel model for the voltage dependence of the forward-running Na+/K+ pump

The voltage dependence of steady state current produced by the forward mode of operation of the endogenous electrogenic Na+/K+ pump in Na(+)- loaded Xenopus oocytes has been examined using a two-microelectrode voltage clamp technique. Four experimental cases (in a total of 18 different experimental conditions) were explored: variation of external [Na+] ([Na]o) at saturating (10 mM) external [K+] ([K]o), and activation of pump current by various [K]o at 0, 15, and 120 mM [Na]o (tetramethylammonium replacement). Ionic current through K+ channels was blocked by Ba2+ (5 mM) and tetraethylammonium (20 mM), thereby allowing pump-mediated current to be measured by addition or removal of external K+. Control measurements and corrections were made for pump current run-down and holding current drift. Additional controls were done to estimate the magnitude of the inwardly directed pump-mediated current that was present in K(+)-free solution and the residual K(+)- channel current. A pseudo two-state access channel model is described in the Appendix in which only the pseudo first-order rate coefficients for binding of external Na+ and K+ are assumed to be voltage dependent and all transitions between states in the Na+/K+ pump cycle are assumed to be voltage independent. Any three-state or higher order model with only two oppositely directed voltage-dependent rate coefficients can be reduced to an equivalent pseudo two-state model. The steady state current-voltage (I-V) equations derived from the model for each case were simultaneously fit to the I-V data for all four experimental cases and yielded least-squares estimates of the model parameters. The apparent fractional depth of the external access channel for Na+ is 0.486 +/- 0.010; for K+ it is 0.256 +/- 0.009. The Hill coefficient for Na+ is 2.18 +/- 0.06, and the Hill coefficient for K+ (which is dependent on [Na]o) ranges from 0.581 +/- 0.019 to 1.35 +/- 0.034 for 0 and 120 mM [Na]o, respectively. The model provides a reasonable fit to the data and supports the hypothesis that under conditions of saturating internal [Na+], the principal voltage dependence of the Na+/K+ pump cycle is a consequence of the existence of an external high- field access channel in the pump molecule through which Na+ and K+ ions must pass in order to reach their binding sites.     

The Gibbs-Donnan near-equilibrium system of heart

The gradients of the major inorganic ions across the plasma membrane of heart were examined to determine the factors controlling the extent and direction of the changes induced during injury, certain diseases, and electrolyte disturbances. The ionic environment was altered by changing only the concentration of inorganic phosphate, [sigma Pi]o, from 0 to 1.2 to 5 mM in the Krebs-Henseleit buffer perfusing working rat hearts. Raising [sigma Pi]o from 1.2 to 5 mM resulted in a decrease in total Mg2+ content and calculated free cytosolic [Mg2+] from 0.44 to 0.04 mM, conversion of 4 mmol of MgATP2- to ATP4- and a decrease in measured intracellular [Cl-]i from 41 to 16 mM. At all levels of [sigma Pi]o, both the [Na+]i and [K+]i were invariant at about 3 mM and 130 mM, respectively, as was the energy of hydrolysis of the terminal phosphate bond of sigma ATP, delta GATP Hydr, of -13.2 kcal/mol. The relationship maintained between the ions on both sides of the plasma membrane by the 3Na+/2K(+)transporting ATPase (EC 3.6.1.37) and an open K+ channel was: (formula; see text) The energy of the gradients of the other inorganic ions across the plasma membrane, delta G[ion]o/i, exhibited three distinct quanta of energy derived from the prime quantum of delta GATP Hydr of -13.2 kcal/mol. The second quantum was about one-third of delta GATP Hydr or +/- 4.4 kcal/mol and comprised the delta G[Na+]o/i, delta G[Mg2+]o/i, and delta G[HPO42-]o/i. These results indicated near-equilibrium was achieved by the reactants of the 3Na+/2K(+)-ATPase, the K+ channel, the Na(+)-Pi co-transporter, and a postulated net Mg2+/H2PO4- exchanger. The third quantum was one-third of delta G[Na+]o/i or about +/- 1.5 kcal/mol and comprised delta G[H+]o/i, delta G[HCO3-]o/i, and delta G[Cl-]o/i. The delta G[K+]o/i was 0, indicating near-equilibrium between the chemical energy of [K+]o/i and the E across the plasma membrane of -83 mV. It is concluded that the gradients of the major inorganic ions across the plasma membrane and the potential across that membrane constitute a Gibbs-Donnan equilibrium system catalyzed by transport enzymes sharing common substrates. The chemical and electrical energies of those gradients are equal in magnitude and opposite in sign to the chemical energy of ATP hydrolysis.     

Controversy regarding the secondary active water transport hypothesis.

Historically, water transport across biological membranes has always been considered a passive process, i.e., the net water transport is proportional to the gradients of hydrostatic and osmotic pressure. More recently, this dogma was challenged by the suggestion that secondary active transporters such as the Na/glucose cotransporter (SGLT1) could perform secondary active water transport with a fixed stoichiometry. In the case of SGLT1, the stoichiometry would consist of one glucose molecule to two Na+ ions to 220-400 water molecules. In the present minireview, we summarize and criticize the evidence supporting and opposing this water cotransport hypothesis. Published and unpublished observations from our own laboratory are also presented in support of the idea that transport-dependent osmotic gradients begin to build up immediately after cotransport commences and are fully responsible for the cell swelling observed.     

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na(+)/glucose cotransporter (SGLT1) using Na(+)- and H(+)-selective electrodes and with the electroneutral H(+)/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na(+)/glucose influx, which generated a measurable decrease in the trap Na(+) concentration ([Na(+)](T)) by 0.080 +/- 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na(+)](T), which was proportional to the increased cotransport current. In a Na(+)-free, weakly buffered solution (pH 5.5), H(+) drives glucose transport through SGLT1, and this was monitored with a H(+)-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na(+)](T) or [H(+)](T) were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na(+)](T) or [H(+)](T) is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.     

The use of HgCl2 to evaluate the cosubstrate: amino acid transport stoichiometry in Ehrlich ascites tumor cells

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and alpha-aminoisobutyric acid (alpha-AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+-free HEPES-buffered medium (pH 7.2) containing 160 mM K+ and 2.5 microM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H-AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, -7.0 +/- 0.1 mV (SEM), does not change with the addition of AIB, -7.3 +/- 0.6 mV (SEM). HgCl2 (10 microM) added to the medium inhibited AIB flux and AIB-stimulated Na+ flux by 45-50% but did not change the coupling ratio. HgCl2 (10 microM) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is -22.3 +/- 0.8 mV (SEM) and depolarizes to -16.7 +/- 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 +/- 0.02 (SEM) in the presence of HgCl2 (10 microM). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of alpha-AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+:AIB stoichiometry approaches 1:1.     

Role of Cl- in electrogenic Na+-coupled cotransporters GAT1 and SGLT1

We have investigated the functional role of Cl(-) in the human Na(+)/Cl(-)/gamma-aminobutyric acid (GABA) and Na(+)/glucose cotransporters (GAT1 and SGLT1, respectively) expressed in Xenopus laevis oocytes. Substrate-evoked steady-state inward currents were examined in the presence and absence of external Cl(-). Replacement of Cl(-) by gluconate or 2-(N-morpholino)ethanesulfonic acid decreased the apparent affinity of GAT1 and SGLT1 for Na(+) and the organic substrate. In the absence of substrate, GAT1 and SGLT1 exhibited charge movements that manifested as pre-steady-state current transients. Removal of Cl(-) shifted the voltage dependence of charge movements to more negative potentials, with apparent affinity constants (K(0.5)) for Cl(-) of 21 and 115 mm for SGLT1 and GAT1, respectively. The maximum charge moved and the apparent valence were not altered. GAT1 stoichiometry was determined by measuring GABA-evoked currents and the unidirectional influx of (36)Cl(-), (22)Na(+), or [(3)H]GABA. Uptake of each GABA molecule was accompanied by inward movement of 2 positive charges, which was entirely accounted for by the influx of Na(+) in the presence or absence of Cl(-). Thus, the GAT1 stoichiometry was 2Na(+):1GABA. However, Cl(-) was transported by GAT1 because the inward movement of 2 positive charges was accompanied by the influx of one Cl(-) ion, suggesting unidirectional influx of 2Na(+):1Cl(-):1GABA per transport cycle. Activation of forward Na(+)/Cl(-)/GABA transport evoked (36)Cl(-) efflux and was blocked by the inhibitor SKF 89976A. These data suggest a Cl(-)/Cl(-) exchange mechanism during the GAT1 transport cycle. In contrast, Cl(-) was not transported by SGLT1. Thus, in both GAT1 and SGLT1, Cl(-) modulates the kinetics of cotransport by altering Na(+) affinity, but does not contribute to net charge transported per transport cycle. We conclude that Cl(-) dependence per se is not a useful criterion to classify Na(+) cotransporters.     

Sugar binding residue affects apparent Na+ affinity and transport stoichiometry in mouse sodium/glucose cotransporter type 3B

SGLT1 is a sodium/glucose cotransporter that moves two Na(+) ions with each glucose molecule per cycle. SGLT3 proteins belong to the same family and are described as glucose sensors rather than glucose transporters. Thus, human SGLT3 (hSGLT3) does not transport sugar, but extracellular glucose depolarizes the cell in which it is expressed. Mouse SGLT3b (mSGLT3b), although it transports sugar, has low apparent sugar affinity and partially uncoupled stoichiometry compared with SGLT1, suggesting that mSGLT3b is also a sugar sensor. The crystal structure of the Vibrio parahaemolyticus SGLT showed that residue Gln(428) interacts directly with the sugar. The corresponding amino acid in mammalian proteins, 457, is conserved in all SGLT1 proteins as glutamine. In SGLT3 proteins, glutamate is the most common residue at this position, although it is a glycine in mSGLT3b and a serine in rat SGLT3b. To test the contribution of this residue to the function of SGLT3 proteins, we constructed SGLT3b mutants that recapitulate residue 457 in SGLT1 and hSGLT3, glutamine and glutamate, respectively. The presence of glutamine at residue 457 increased the apparent Na(+) and sugar affinities, whereas glutamate decreased the apparent Na(+) affinity. Moreover, glutamate transported more cations per sugar molecule than the wild type protein. We propose a model where cations are released intracellularly without the release of sugar from an intermediate state. This model explains the uncoupled charge:sugar transport phenotype observed in wild type and G457E-mSGLT3b compared with SGLT1 and the sugar-activated cation transport without sugar transport that occurs in hSGLT3.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Molecular mechanism of iodide transport by thyroid plasmalemmal vesicles: cooperative sodium activation and asymmetrical affinities for the ions on the outside and inside of the vesicles

The 125I- uptake by plasmalemmal vesicles from porcine thyroid was measured by a Millipore filtration method using 2 mM ClO4- as a reaction stopper. Effective uptake occurred in the presence of high concentrations of extravesicular Na+ (Na+o). In the presence of Na-ionophores such as monensin and nigericin, no uptake was observed and the accumulated I- was released. The initial rate of I- uptake increased with the concentration of extravesicular I- (I-o) according to simple saturation kinetics and [I-o] giving a half-maximum rate of about 5 microM. The dependence of the rate on [Na+o] showed cooperativity with a Hill coefficient of 1.8, and a KNa value of 0.0064 M2, suggesting that the binding of at least 2 Na+ ions to a carrier molecule was required to transport an I- ion. Further kinetic data were consistent with a mechanism in which bindings of the ions were rapid and the Na+ binding occurred prior to the I- binding. Intravesicular Na+ inhibited the I- uptake and the inhibition constant (KiNa) was about 4 mM, independently of [I-o] and [Na+o]. Intravesicular I- inhibited the I- uptake with an apparent KiI value of about 100 microM. The results suggest that the differences in the Na+- and I- -binding modes between outside and inside of the vesicles are important factors causing the I- uptake against its concentration gradient.     

Occlusion of 22Na+ and 86Rb+ in membrane-bound and soluble protomeric alpha beta-units of Na,K-ATPase

In this work, we examined occlusion of 22Na+ and 86Rb+ in membranous and detergent-solubilized Na,K-ATPase from outer renal medulla. Optimum conditions for occlusion of 22Na+ were provided by formation of the phosphorylated complex from the beta,gamma-bidentate complex of chromium (III) with ATP (CrATP). Release of occluded cations occurred at equally slow rates in soluble and membrane-bound Na,K-ATPase. Values of 22Na+ occlusion as high as 11 nmol/mg of protein were measured, corresponding to 1.8-2.7 mol of Na+/mol of phosphorylated Na,K-ATPase as determined by 32P incorporation from [gamma-32P]CrATP. Maximum capacity for phosphorylation from [gamma-32P]CrATP was 6 nmol/mg of protein and equal to capacities for binding of [48V]vanadate and [3H]ouabain. The stoichiometry for occlusion of Rb+ was close to 2 Rb+ ions/phosphorylation site. In an analytical ultracentrifuge, the soluble Na+- or Rb+-occluded complexes showed sedimentation velocities (S20,w = 6.8-7.4) consistent with monomeric alpha beta-units. The data show that soluble monomeric alpha beta-units of Na,K-ATPase can occlude Rb+ or Na+ with the same stoichiometry as the membrane-bound enzyme. The structural basis for occlusion of cations in Na,K-ATPase is suggested to be the formation of a cavity inside a monomeric alpha beta-unit constituting the minimum protein unit required for active Na,K-transport.     

Voltage-dependent modulation of ion binding and translocation in the cardiac Na(+)-Ca2+ exchange system

The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).     

Effect of Extracellular Potassium on Ouabain-Sensitive Consumption of High-Energy Phosphate by Crayfish Giant Axons: A Study of the Energy Requirement for Transport in the Steady State

Crayfish axons exposed to a high or low extracellular K+ concentration ([K+]o) maintain intracellular Na+ and K+ concentrations constant, for up to 3 h, by adjusting both the Na+/K+ transport "coupling ratio" and turnover rate in compensation for changes in ion fluxes due to altered electrochemical gradients. These findings give rise to the prediction that the steady-state consumption of high-energy phosphate (approximately P) [ATP and phospho-L-arginine (Arg-P)] is inversely proportional to the [K+]o, i.e., directly proportional to the product of membrane conductance and magnitude of the transmembrane electrochemical gradients for Na+ and K+. This investigation was designed to test this hypothesis. The [K+]o did not influence total approximately P consumption (Q approximately P) of the axon. For a [K+]o between 0.5 and 21.6 mM, Q approximately P averaged 52.8 +/- 4.7%/h (n = 44) of the initial [ATP] + [Arg-P]. Unlike total Q approximately P, the ouabain-sensitive portion of Q approximately P was markedly influenced by [K+]o. In 0.5 mM K+o, ouabain poisoning reduced Q approximately P to 8%/h, a result indicating that 85% of the total Q approximately P was ouabain sensitive. For 1.35 mM K+o, the ouabain-sensitive portion was 66%; at 5.4 mM K+o, 45%; and at 13.5 mM K+o, 41%. There was a small but significant increase in the ouabain-sensitive Q approximately P at 21.6 mM K+o, compared with Q approximately P at 5.4 mM K+o. The pattern of effect of [K+]o on Q approximately P was similar to its effect on the electrical power content of the Na+ and K+ electrochemical gradients. In contrast to the generally accepted Na+ flux (JNa)/approximately P stoichiometry of 3, an actual ratio of JNa/approximately P stoichiometry of approximately 33:1 was calculated for the experiments reported here, a result suggesting that cells in a zero-membrane current steady state utilize efficient energy conservation mechanisms that may not operate under non-steady-state conditions.     

Stoichiometry of the Cardiac Na+/Ca2+ exchanger NCX1.1 measured in transfected HEK cells

The stoichiometry with which the Na+/Ca2+ exchanger, NCX1, binds and transports Na+ and Ca2+ has dramatic consequences for ionic homeostasis and cellular function of heart mycocytes and brain neurons, where the exchanger is highly expressed. Previous studies have examined this question using native NCX1 in its endogenous environment. We describe here whole-cell voltage clamp studies using recombinant rat heart NCX1.1 expressed heterologously in HEK-293 cells. This system provides the advantages of a high level of NCX1 protein expression, very low background ion transport levels, and excellent control over clamped voltage and ionic composition. Using ionic conditions that allowed bi-directional currents, voltage ramps were employed to determine the reversal potential for NCX1.1-mediated currents. Analysis of the relation between reversal potential and external [Na+] or [Ca2+], under a variety of intracellular conditions, yielded coupling ratios for Na+ of 1.9-2.3 ions per net charge and for Ca2+ of 0.45 +/- 0.03 ions per net charge. These data are consistent with a stoichiometry for the NCX1.1 protein of 4 Na+ to 1 Ca2+ to 2 charges moved per transport cycle.     

Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.

We have used baculovirus (AcNPV) to express the Na+/glucose cotransporter protein in cultured Sf9 cells. We constructed a baculovirus transfer vector containing the cDNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) under the control of the polyhedrin gene promoter. Recombinant baculovirus was obtained by cotransfection of SF9 cells with wild-type AcNPV DNA and the transfer vector. Recombinant virus was identified by Southern blotting and then purified. Recombinant infected Sf9 cells expressed a protein which was recognized by anti-peptide antibodies raised to sequences of the cloned Na+/glucose cotransporter. This protein migrated with a molecular mass of 55 kD by SDS-PAGE, similar to the in vitro translation product of SGLT1. An identical protein was metabolically labeled with [35S]methionine. Cells which synthesized the transport protein showed Na(+)-dependent alpha MeGlc transport. Micromolar phlorizin inhibited transport. Uninfected and wild-type virus infected Sf9 cells did not have Na(+)-dependent glucose transport. All transport protein migrated at 45% sucrose (w/w) by density gradient sedimentation, suggesting that the expressed transporter is membrane associated. We conclude that we have functionally expressed the rabbit intestinal Na+/glucose cotransporter in Sf9 cells. The transporter is not heavily glycosylated, and this is consistent with previous work showing that glycosylation is not necessary for function. We are poised to purify and characterize this protein from a structure-function perspective.     

Na(+)-dependent Cl-HCO3 exchange in the squid axon. Dependence on extracellular pH.

Intracellular pH (pHi) in squid giant axons recovers from acid loads by means of a Na(+)-dependent Cl-HCO3 exchanger, the actual mechanism of which might be exchange of: (i) external Na+ and HCO3- for internal Cl- and H+, (ii) Na+ plus two HCO3- for Cl-, (iii) Na+ and CO3= for Cl-, or (iv) the NaCO3- ion pair for Cl-. Here we examine sensitivity of transport to changes of extracellular pH (pHo) in the range 7.1-8.6. We altered pHo in four ways, using: (i) classical "metabolic" disturbances in which we varied [HCO3-]o, [NaCO3-]o, and [CO3=]o at a fixed [CO2]o; (ii) classical "respiratory" disturbances in which we varied [CO2]o, [NaCO3-]o, and [CO3=]o at a fixed [HCO3-]o; (iii) novel mixed-type acid-base disturbances in which we varied [HCO3-]o and [CO2]o at a fixed [CO3=]o and [NaCO3-]o; and (iv) a second series of novel mixed-type disturbances in which we varied [CO2]o, [CO3=]o, and [Na+]o at a fixed [HCO3-]o and [NaCO3-]o. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. The equivalent acid extrusion rate (JH) was computed from the rate of pHi recovery (i.e., increase) in the presence of Na+ and HCO3-. When pHo was varied by method (i), which produced the greatest range of [CO3=]o and [NaCO3-]o values, JH increased with pHo in a sigmoidal fashion; the relation was fitted by a pH titration curve with a pK of approximately 7.7 and a Hill coefficient of approximately 3.0. With method (ii), which produced smaller changes in [CO3=]o and [NaCO3-]o, JH also increased with pHo, though less steeply. With method (iii), which involved changes in neither [CO3=]o nor [NaCO3-]o, JH was insensitive to pHo changes. Finally, with method (iv), which involved changes in neither [HCO3-] nor [NaCO3-]o, but reciprocal changes in [CO3=]o and [Na+]o, JH also was insensitive to pHo changes. We found that decreasing pHo from 8.6 to 7.1 caused the apparent Km for external HCO3- ([Na+]o = 425 mM) to increase from 1.0 to 26.7 mM, whereas Jmax was relatively stable. Decreasing pHo from 8.6 to 7.4 caused the apparent Km values for external Na+ ([HCO3-]o = 48 mM) to increase from 8.6 to 81 mM, whereas Jmax was relatively stable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sodium-calcium exchange current. Dependence on internal Ca and Na and competitive binding of external Na and Ca

Na-Ca exchange current was measured at various concentrations of internal Na [( Na]i) and Ca [( Ca]i) using intracellular perfusion technique and whole-cell voltage clamp in single cardiac ventricular cells of guinea pig. Internal Ca has an activating effect on Nai-Cao exchange beginning at approximately 10 nM and saturating at approximately 50 nM with a half maximum [Ca]i (Km[Ca]i) of 22 nM (Hill coefficient, 3.7). Measurement of Nai-Cao exchange current at various concentration of [Na]i revealed an apparent Km[Na]i of 20.7 +/- 6.9 mM (n = 14) with imax of 3.5 +/- 1.2 microA/microF. For [Ca]i transported by the exchange, a Km[Ca]i of 0.60 +/- 0.24 microM (n = 8) with an imax of 3.0 +/- 0.54 microA/microF was obtained by measuring Nao-Cai exchange current. These values are apparently different from the values for the external binding site which have been reported previously. Whether Na and Ca compete for the external binding site, and if so, how it affects the binding constants was then investigated. Outward Nai-Cao exchange current became larger by reducing [Na]o. The double reciprocal plot of the current magnitude and [Ca]o at different [Na]o revealed a competitive interaction between Na and Ca. In the absence of competitor [Na]o, an apparent Km[Ca]o of 0.14 mM was obtained. When comparing internal and external Km values, the external value is markedly larger than the internal one and thus we conclude that binding sites of the Na-Ca exchange molecule are at least apparently asymmetrical between the inside and outside of the membrane.