The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.     

The effects of monovalent cations Li+, Na+, K+, NH4+, Rb+ and Cs+ on the solid and solution structures of the nucleic acid components. Metal ion binding and sugar conformation.

The interactions of the monovalent ions Li+, Na+, K+, NH4+, Rb+ and Cs+ with adenosine-5'-monophosphoric acid (H2-AMP), guanosine-5'-monophosphoric acid (H2-GMP) and deoxyguanosine-5'-monophosphoric acid (H2-dGMP) were investigated in aqueous solution at physiological pH. The crystalline salts M2-nucleotide.nH2O, where M = Li+, Na+, K+ NH4+, Rb+ and Cs+, nucleotide = AMP, GMP and dGMP anions and n = 2-4 were isolated and characterized by Fourier Transform infrared (FTIR) and 1H-NMR spectroscopy. Spectroscopic evidence showed that these ions are in the form of M(H2O)n+ with no direct metal-nucleotide interaction, in aqueous solution. In the solid state, Li+ ions bind to the base N-7 site and the phosphate group (inner-sphere), while the NH4+ cations are in the vicinity of the N-7 position and the phosphate group, through hydrogen bonding systems. The Na-nucleotides and K-nucleotides are structurally similar. The Na+ ions bind to the phosphate group of the AMP through metal hydration shell (outer-sphere), whereas in the Na2-GMP, the hydrated metal ions bind to the base N-7 or the ribose hydroxyl groups (inner-sphere). The Na2-dGMP contains hydrated metal-carbonyl and metal-phosphate bindings (inner-sphere). The Rb+ and Cs+ ions are directly bonded to the phosphate groups and indirectly to the base moieties (via H2O). The ribose moiety shows C2'-endo/anti conformation for the free AMP acid and its alkali metal ion salts. In the free GMP acid, the ribose ring exhibits C3'-endo/anti conformer, while a C2'-endo/anti sugar pucker was found in the Na2-GMP and K2-GMP salts and a C3'-endo/anti conformation for the Li+, NH4+, Rb+ and Cs+ salts. The deoxyribose has C3'-endo/anti conformation in the free dGMP acid and O4'-endo/anti in the Na2-dGMP, K2-dGMP and a C3'-endo/anti for the Li+, NH4+, Rb+ and Cs+ salts. An equilibrium mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers was found for these metal-nucleotide salts in aqueous solution.     

(Na+ + K+)-adenosine triphosphatase of mammalian brain. Catalytic and regulatory K+ sites distinguishable by selectivity for Li+.

Li+, K+, and Rb+ are compared as activators of the hydrolysis of p-nitrophenylphosphate by beef brain (Na+ + K+)-ATPase. Previous experiments have established two classes of K+ binding sites that are involved in this reaction: "catalytic sites" have the higher affinity, their occupation is essential for catalytic activity, and they appear to correspond to the extracellular binding sites for active K+ transport; regulatory sites appear to have an allosteric function to "unmask" the catalytic sites. A separate set of Na+-binding regulatory sites bring about a similar unmasking of catalytic sites under phosphorylating conditions. Rb+ can activate p-nitrophenylphosphate hydrolysis both in the presence and absence of Na+ and, thus, can interact effectively with both K+ regulatory and catalytic sites. Li+ does not activate p-nitrophenylphosphate hydrolysis at 25 degrees C in the absence of other monovalent ligands. Li+ does activate when the catalytic sites are exposed by Na+ + ATP. Thus, K+ regulatory and catalytic sites differ in their cation selectivity. At temperatures less than 25 degrees C Li+ is able to activate the phosphatase reaction in the absence of other monovalent ligands: maximum activity occurs at 10-12 degrees C. A plot of the ratio, Li+ activation/K+ activation, as a function of temperature shows that the allosteric transition that unmasks catalytic sites occurs spontaneously with decreasing temperatures.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

Complexation studies on inositol-phosphates. II. Alkali-metal complexes of D-myo-inositol 1,2,6 trisphosphate

The complexation properties of the D-myo-inositol 1,2,6 trisphosphate (Ins(1,2,6)P3) towards Li+, Na+, K+, Rb+, and Cs+ cations were studied at 25 degrees C in a 0.1 M tetra-n-butylammonium bromide medium. For all cations, mononuclear and protonated species were found. For smaller cations (Li+, Na+, and K+) a dinuclear complex was also put into evidence. The main characteristic of the complexes is its high stability; and of the ligand, its nonselectivity. The Ins(1,2,6)P3-K system was ascertained using Sammartano's method which additionally enabled the influence of various K+ concentrations on the protonations constants to be considered.     

Temperature dependence of ion permeation at the endplate channel

The dependence of acetylcholine receptor mean single-channel conductance on temperature was studied at garter snake twitch-muscle endplates using fluctuation analysis. In normal saline under conditions where most of the endplate current was carried by Na+, the channel conductance increased continuously from near 0 degrees C to approximately 23 degrees C with a Q10 of 1.97 +/- 0.14 (mean +/- SD). When 50% of the bath Na+ was replaced by either Li+, Rb+, or Cs+, the Q10 did not change significantly; however, at any temperature the channel conductance was greatest in Cs-saline and decreased with the ion sequence Cs greater than Rb greater than Na greater than Li. The results were fit by an Eyring-type model consisting of one free-energy well on the extracellular side of a single energy barrier. Ion selectivity appeared to result from ion-specific differences in the well and not in the barrier of this model. With a constant barrier enthalpy for different ions, well free-energy depth was greatest for Cs+ and graded identical to the permeability sequence. The correlation between increased well depth (i.e., ion binding) and increased channel conductance can be accounted for by the Boltzmann distribution of thermal energy.     

Duplex-tetraplex equilibrium between a hairpin and two interacting hairpins of d(A-G)10 at neutral pH.

d(A-G)10 forms two helical structures at neutrality, at low ionic strength a single-hairpin duplex, and at higher ionic strength a double-hairpin tetraplex. An ionic strength-dependent equilibrium between these forms is indicated by native PAGE, which also reveals additional single-stranded species below 0.3 M Na+, probably corresponding to partially denatured states. The equilibrium also depends upon oligomer concentration: at very low concentrations, d(A-G)10 migrates faster than the random coil d(C-T)10, probably because it is a more compact single hairpin; at high concentrations, it co-migrates with the linear duplex d(A-G)10 x d(C-T)10, probably because it is a two-hairpin tetraplex. Molecular weights measured by equilibrium sedimentation in 0.1 M Na+, pH 7, reveal a mixture of monomer and dimer species at 1 degree C, but only a monomer at 40 degrees C; in 0.6 M Na+, pH 7, only a dimer species is observed at 4 degrees C. That the single- and double-stranded species are hairpin helices, is indicated by preferential S1 nuclease cleavage at the center of the oligomer(s), i.e., the loop of the hairpin(s). The UV melting transition below 0.3 M Na+ or K+, exhibits a dTm/dlog[Na+/K+] of 33 or 36 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex to a single-hairpin duplex with extrahelical residues. When [Na+/K+] > or = 0.3 M, dTm/dlog [Na+/K+] is 19 or 17 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex directly to single strands. A two-hairpin structure stabilized by G-tetrads is indicated by differential scanning calorimetry in 0.15 M Na+/5 mM Mg2+, with deltaH of formation per mole of the two-hairpin tetraplex of -116.9 kcal or -29.2 kcal/mol of G-tetrad.     

Insulin activation of (Na+,K+)-adenosinetriphosphatase exhibits a temperature-dependent lag time. Comparison to activation of the glucose transporter

The time course of insulin activation of sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)ATPase] was studied in the rat adipocyte and was compared to activation of the glucose transporter. Under conditions in which the binding of insulin to its cell surface receptor was not rate limiting, a distinct time lag was apparent between insulin addition and stimulation of transport activity. At 37 degrees C, 40-50 s elapsed before an increase in Rb+ uptake [a measure of (Na+,K+)ATPase transport activity] or 2-deoxyglucose uptake could be observed. This lag time increased in an identical manner for both transport processes as the temperature was lowered to 23 degrees C. Addition of the insulinomimetic agent hydrogen peroxide also produced a lag time similar to that for insulin before activation of Rb+ and 2-deoxyglucose uptakes was detected. These data provide the first evidence of a discrete time lag involved during stimulation of the adipocyte (Na+,K+)ATPase. A model for the molecular mechanism of insulin activation of (Na+,K+)ATPase is presented that incorporates these results into the hypothesis of insulin mediated "translocation" of glucose transporters to the plasma membrane.     

Transport of univalent cations across the erythrocyte membrane of hypertensive rats of various ages

In the erythrocytes incubated at low temperature (3-6 degrees C), the uptake of Li+ in 6- and 16-week old spontaneously hypertensive rats (SHR) was significantly higher than in the normotensive rats (WKY) of the same age. During the incubation of cells at 37 degrees C no difference occurred in either ouabain-sensitive or ouabain-resistant fluxes of Rb+, Na+ and Li+ between the 16-week old SHR and the WKY. K+ efflux from the erythrocytes at 3 degrees C was consistently stimulated after addition to the incubation medium of 1 mmol/l Ca2+. The value of Ca2+-dependent K+-transport was significantly elevated in 16-week old SHR than in the WKY, but there was no difference in 6-week old rats. Propranolol-induced Ca2+-dependent K+ efflux from the cells at 22 degrees C was markedly higher in 6- and 16-week old SHR as compared with the WKY. The results provide a further evidence in favor of the hypothesis on the existence of a "membrane defect" in red blood cells in the SHR.     

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.     

Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP

We have measured the time course of release of 42K and 86Rb from an occluded state of the Na,K-pump using a rapid filtration apparatus. We have found that at 20 degrees C and in the presence of ATP, 42K is released with a rate constant of approximately 45 s-1 and 86Rb with a rate constant of approximately 20 s-1; both ATP and ADP are effective at a low affinity site (Kd approximately 0.3 and 1 mM, respectively) with the rate of deocclusion being only half as great in ADP as in ATP. Mg2+ stimulates 2-fold at low concentrations probably by forming MgATP, and free Mg2+ is strongly inhibitory at high concentrations (Kd approximately 10 mM). Mg2+ also decreases the affinity for ATP, and the data are consistent with mixed type inhibition; from the analysis the dissociation constant is approximately 1 mM for the inhibitory Mg2+ and the Rb+-occluded form without ATP. The rate of 42K or 86Rb release increases monotonically with pH while ATPase activity decreases above pH 8, so that deocclusion is not rate-limiting in the overall cycle at high pH. This is reflected by a convergence of the rate of Na,K-ATPase and Na,Rb-ATPase activities at high pH and by a decrease in the observed steady-state level of the occluded 86Rb intermediate at high pH. K+, Rb+, Na+, and Cs+, but not Li+, increase the rate of 42K and 86Rb release at constant ionic strength, presumably at sites other than the transport sites. The spontaneous rate of deocclusion is only approximately 0.1 s-1 at low ionic strength in the absence of nucleotides, and it is increased markedly by all cations tested except Li+. Overall the data are consistent with deocclusion as a rate-limiting step in the Na,K-pump cycle.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

The permeability of aconitine-modified sodium channels to univalent cations in myelinated nerve

1. Ionic currents through the sodium system of nodes of Ranvier treated with aconitine were measured under voltage clamp conditions in a Ringer solution containing Na+ or an equimolar amount of various test cations. 2. Average shifts in reversal potentials in nodes of Ranvier treated with aconitine with NH4+, Li+, K+, Rb+, Cs+ in place of Na+ in the Ringer solution are 7.6, --6.8, --25.0, --41.0 and --51.5 mV at 13--14degrees C. At 20--22degrees C the sequence of shifts is 7.5, --5.5, --13.5, --29.0 and --41.0 mV. For Tl+ the the average reversal potential shift is +3 mV at 20--22degrees C. 3. The slope of the instantaneous current-voltage relation at the reversal potential in nodes treated with aconitine changed with the various cations tested. The ratios are NH4+/Na+/K+/Rb+/Cs+/Li+ = 1.14 : 1.0 : 0.80 :0.67 :0.53 : 0.53. 4. Using a three energy barrier model some of the parameters for the aconitine-modified Na+ channels were estimated (Chizmadgev, Yu. A., Khodorov, B.I. and Aityan, S.Kh. (1974) Bioelectrochem. Bioenerg. 1, 301--312).     

Potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a Na+, K+, Cl- cotransporter by protein kinase C.

The mechanisms by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain-inhibitable bumetanide-insensitive 86Rb+ transport accounted for approximately 70-80% of total, whereas bumetanide-inhibitable ouabain-insensitive uptake accounted for 15-25% of total. K+ channel blockers such as BaCl2 reduced uptake by approximately 5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 microM, while furosemide inhibited with an IC50 of about 20 microM. Bumetanide-inhibitable 86Rb+ uptake was reduced in Na(+)-free or Cl(-)-free media, suggesting that Na+ and Cl- were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl- cotransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, caused a 50-70% decrease in 86Rb+ uptake via the Na+, K+, Cl- cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl- cotransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl- cotransporter in NPE cells is inhibited by activation of protein kinase C.     

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guinea pig jejunal brush border membrane vesicles

D-Glucose transport was studied with isolated brush border membrane vesicles from guinea pig jejunum. Saturation curves were carried out at either 25 or 35 degrees C in buffers containing Na+, Li+, K+ (100 mM chloride salt), or sorbitol (200 mM). Uncorrected uptake rates were fitted by nonlinear regression analysis to an equation involving one diffusional and two saturable terms. In the presence of Na+ at 35 degrees C, two saturable systems (Km = 0.4 and 24 mM, respectively) were evident, as well as a diffusion component quantitatively identical with that measured with L-glucose in separate experiments. In contrast, at 25 degrees C only one saturable system was apparent (Km = 1.2 mM): the second exhibited diffusion-like kinetics. In the presence of Na+ at 35 degrees C, D-glucose uptake was fully inhibited by both D-glucose and D-galactose, whereas alpha-methylglucoside gave kinetics of partial inhibition. We conclude that in the presence of Na+ there are at least two distinct D-glucose transport systems: 1) System I, a low temperature-sensitive system, fully inhibited by D-glucose, D-galactose, and alpha-methylglucoside; we identify it as the "classical" D-glucose/Na+ cotransport system, insensitive to inhibition by cytochalasin B and obligatorily dependent on Na+; and 2) System II, a high temperature-sensitive system where D-glucose and D-galactose inhibit but alpha-methylglucoside is inert. Its cation specificity is unclear but it appears to be sensitive to cytochalasin B inhibition. When Li+ or K+ substituted for Na+, only one transport system was apparent. The Li+-activated transport was: independent of the incubation temperature; inhibited by D-glucose and D-galactose but not by alpha-methylglucoside, 2-deoxy-D-glucose, D-mannose, and D-xylose; and sensitive to cytochalasin B inhibition. The exact nature of the system (or systems) involved in D-glucose transport in the absence of sodium remains to be established.     

Microsomal (Na- +K+)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors.

A method is described for the extraction of microsomal ouabain-sensitive (a- + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ K+)-stimulated activity in the range of 30- 40 nmol - mg -1 - min -1 at 26 degrees C. This portion which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+. The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump.     

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.