Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

Gastric (H,K)-ATPase

Gastric acid secretion results from the activity of a specific ATPase, the (H+,K+)-ATPase. This enzyme, discovered in 1973, exchanges H+ for K+. It has two ATP binding sites, both involved in enzyme activity, whose affinities vary as a function of the H+ and K+ concentrations. Hydrolysis of ATP at the highest affinity site leads to the synthesis of a covalent aspartyl phosphate which accumulates in the absence of K+. The presence of this cation accelerates dephosphorylation resulting in the stimulation of ATPase (and PNPPase) activity. The structure of membranous (H+,K+)-ATPase is poorly defined. n-Octylglucoside solubilizes an active enzyme of 390-420 kDa which can be partly depolymerized using cholate. The monomer, characterized in SDS has a 95 kDa molecular mass and is inactive. In the presence of magnesium, (H+,K+)-ATPase catalyzes the active and neutral exchange of H+ for K+ at the expense of ATP. In the absence of ATP, (H+,K+)-ATPase acts as a passive transporter exchanging K+ for K+ at maximal rate and H+ for K+ at a 20 times slower rate.     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.     

Importance of Leu99 in transmembrane segment M1 of the Na+, K+ -ATPase in the binding and occlusion of K+

Twenty-six point mutations were introduced into the N-terminal and middle parts of transmembrane segment M1 of the Na+, K+ -ATPase and its cytosolic extension. None of the alterations to charged and polar residues in the N-terminal part of M1 and its cytosolic extension had any major effect on the cation binding properties, thus rejecting the hypothesis that these residues are involved in cation selectivity. By contrast, specific residues in the middle part of M1, particularly Leu(99), were found critical to K+ interaction of the enzyme. Hence, mutation L99A reduced the affinity for K+ activation of E2P dephosphorylation 17-fold, and L99F reduced the equilibrium level of the K+-occluded intermediate [K2]E2 and increased the rate of K+ deocclusion 39-fold, i.e. more than seen for mutation E329Q of the cation-binding glutamate in M4. L99Q affected K+ interaction in yet another way, the equilibrium level of [K2]E2 being slightly increased despite an increased rate of K+ deocclusion, suggesting that the K+ ions leave and enter the occlusion pocket more frequently than in the wild type. L99Q furthermore affected the ability to discriminate between Na+ and K+ on the extracellular side. Our findings can be explained by a structural model in which Leu(99) and Glu(329) interact and cooperate in K+ binding and gating of the K+ sites. The disturbance of K+ interaction in mutants with alteration to Leu(91), Phe(95), Ser(96), or Leu(98) could be a consequence of the roles of these residues in positioning the M1 helix optimally for the interaction between Leu(99) and Glu(329). Phe(95) may serve to stabilize the pivot for movement of M1 through interaction with Ile(287) in M3.     

Functional Consequences of Various Leucine Mutations in the M3/M4 Loop of the Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase α-Subunit

Leucines were mutated within the sequence L³¹¹ILGYTWLE³¹⁹ of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na⁺,K⁺-ATPase α-subunit. Replacement of Leu³¹¹ with Glu resulted in a considerable loss of Na⁺,K⁺-ATPase activity. Replacement of Leu³¹³ with Glu shifted the equilibrium of E₁P and E₂P toward E₁P and reduced the rate of the E₁P to E₂P transition. The reduction of the transition rate and stronger inhibition of Na⁺,K⁺-ATPase activity by Na⁺ at higher concentrations together suggest that there is interference of Na⁺ release on the extracellular side in the Leu³¹³ mutant. Thus, Leu³¹³ could be in the pathway of Na⁺ exit. Replacement of Leu³¹⁸ with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K⁺, with an equilibrium shifted toward E₂P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K⁺ ₂]E₂ caused by inhibition of dephosphorylation through reduction of the K⁺ affinity of E₂P. Thus, Leu³¹⁸ may be a critical position in guiding external K⁺ to its binding site.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Trp17 and Glu20 residues in conserved WMN(D/E)PN motif are essential for Aspergillus ficuum endoinulinase (EC 3.2.1.7) activity

The importance of the WMN(D/E)PN motif, which is well conserved among -fructofuranosidases grouped in the glycosylhydrolase family 32, in Aspergillus ficuum endoinulinase was accessed. Each mutant enzyme generated by site-directed mutagenesis of Trp17 in the conserved motif to Gln, Leu, Ser, Pro, Thr, or Met had an activity of less than 1% of the wild type. Another mutant enzyme obtained by mutation of Glu20 in the motif to Ser, Leu, Thr, Gln, Ala, or Val had an enzyme activity of less than 1% of the wild type. Furthermore, the E20D mutant enzyme, in which Glu20 in the conserved motif was replaced with Asp, had 1.1% of the wild type activity. These results clearly indicated that Trp17 and Glu20 are essential for the enzyme activity.     

Diclofenac-induced peripheral antinociception is associated with ATP-sensitive K+ channels activation

In order to investigate to the contribution of K+ channels on the peripheral antinociception induced by diclofenac, we evaluated the effect of several K+ channel blockers, using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 microg) of prostaglandin E2. Diclofenac administered locally into the right hindpaw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. This blockade of PGE2 mechanical hyperalgesia induced by diclofenac (100 microg/paw) was antagonized in a dose-dependent manner by intraplantar administration of the sulphonylureas glibenclamide (40, 80 and 160 microg) and tolbutamide (80, 160 and 320 microg), specific blockers of ATP-sensitive K+ channels, and it was observed even when the hyperalgesic agent used was carrageenin, while the antinociceptive action of indomethacin (200 microg/paw), a typical cyclo-oxygenase inhibitor, over carrageenin-induced hyperalgesia was not affected by this treatment. Charybdotoxin (2 microg/paw), a blocker of large conductance Ca2+-activated K+ channels and dequalinium (50 microg/paw), a selective blocker of small conductance Ca2+-activated K+ channels, did not modify the effect of diclofenac. This effect was also unaffected by intraplantar administration of non-specific voltage-dependent K+ channel blockers tetraethylammonium (1700 microg) and 4-aminopyridine (100 microg) or cesium (500 microg), a non-specific K+ channel blocker. The peripheral antinociceptive effect induced by diclofenac was antagonized by NG-Nitro L-arginine (NOarg, 50 microg/paw), a NO synthase inhibitor and methylene blue (MB, 500 microg/paw), a guanylate cyclase inhibitor, and this antagonism was reversed by diazoxide (300 microg/paw), an ATP-sensitive K+ channel opener. We also suggest that an endogenous opioid system may not be involved since naloxone (50 microg/paw) did not affect diclofenac-induced antinociception in the PGE2-induced hyperalgesia model. This study provides evidence that the peripheral antinociceptive effect of diclofenac may result from activation of ATP-sensitive K+ channels, possible involving stimulation of L-arginine/NO/cGMP pathway, while Ca2+-activated K+ channels, voltage-dependent K+ channels as well as endogenous opioids appear not to be involved in the process.     

Dichotomic phylogenetic tree of the pyruvate kinase family: K+ -dependent and -independent enzymes

K+ dependence was assumed to be a feature of all pyruvate kinases until it was discovered that some enzymes express K+ -independent activity. Almost all the K+ -independent pyruvate kinases have Lys at position 117, instead of the Glu present in the K+ -dependent muscle enzyme. Mutagenesis studies show that the internal positive charge substitutes for the K+ requirement (Laughlin, L. T. & Reed, G. H. (1997) Arch. Biochem. Biophys. 348, 262-267). In this work a phylogenetic analysis of pyruvate kinase was performed to ascertain the abundance of K+ -independent activities and to explore whether the K+ activating effect is related to the evolutionary history of the enzyme. Of the 230 studied sequences, 46% have Lys at position 117, and the rest have Glu. Pyruvate kinases with Lys117 and Glu117 are separated in two clusters. All of the enzymes of the Glu117 cluster that have been characterized are K+ -dependent, whereas those of the Lys117 cluster are K+ -independent. Thus, there is a strict correlation between the dichotomy of the tree and the dependence of activity on K+. 77% of the pyruvate kinases that possess Lys117 have Lys113/Gln114; they also have Ile, Val, or Leu at position 120. These residues are replaced by Glu117 and Thr113/Lys114/Thr120 in 80% of K+ -dependent pyruvate kinases. Structural analysis indicates that these residues are in a hinge region involved in the acquisition of the catalytic conformation of the enzyme. The route of conversion from K+ -independent to K+ -dependent pyruvate kinases is described. A plausible explanation of how enzymes developed K+ dependence is put forth.     

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase

Goat antisera against (Na⁺ + K⁺)-ATPase and its isolated subunits and against (K⁺ + H⁺)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na⁺ + K⁺)-ATPase antisera cross-reacted with (K⁺ + H⁺)-ATPase or inhibited its enzyme activity. The same was true for the (K⁺ + H⁺)-ATPase antiserum with regard to (Na⁺ + K⁺)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na⁺ + K⁺)-Activated ATPase.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

Urea inhibition of renal (NA+ + K+)ATPase activity is reversed by cAMP

In the present work we studied the modulation of the effect of urea on the renal (Na+ + K+)ATPase by cAMP. We observed that urea inhibits the (NA+ + K+)ATPase activity in a dose-dependent manner, reaching 60% of inhibition at the concentration of 1M. This effect was completely reversed by dibutyryl-cAMP (dBcAMP) at 5 x 10(-4)M. The effect of dBcAMP was mimicked by 50 units of the catalytic subunit of protein kinase A and completely abolished by 5 x 10(-7)M H89, an inhibitor of protein kinase A. Addition of 1M urea decreases basal phosphorylation of the immunoprecipitated (NA+ + K+)ATPase in 50%, with this effect completely reversed by 5 x 10(-4)M dBcAMP. Furthermore, 5 x 10(-4)M dBcAMP by itself induced (NA+ + K+)ATPase phosphorylation. Taken together these data indicate that cAMP could be, in addition to the organic solutes already known, an important physiological modulator of the deleterious effect of urea on enzyme activity.     

Stimulation of Na+,K+-ATPase activity in certain membranes of the rat central nervous system (CNS) by acute administration of desipramine (DMI)

1. The activities of ATPase in rat CNS were studied 3 hr after administration of the noradrenaline uptake inhibitor, desipramine (DMI: 10 mg.kg-1, i.p.). Na+K+-ATPase activity significantly increased after DMI in the whole particulate from hypothalamus and mesencephalus but no changes in frontal cortex or in pons-medulla oblongata areas were found. This increase was prevented when the animals were pretreated with the noradrenergic neurotoxic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). 2. Purified membrane fractions from hypothalamus were obtained by differential and sucrose gradient centrifugation (0.8-1.2 M sucrose). It was observed that after DMI, Na+,K+-ATPase activity increased only in the membranous fraction lying at 0.9 M sucrose. 3. Mg2+- or Ca2+-ATPase activities were not modified by DMI treatment. 4. Citalopram, a specific serotonergic uptake inhibitor, did not affect ATPase activities. 5. The results obtained could indicate that DMI acute administration selectively stimulates Na+,K+-ATPase activity of certain membranes of the CNS after an increase in the concentration of the noradrenergic neurotransmitter in the synaptic gap.     

Characterization of K+-dependent and K+-independentp-nitrophenylphosphatase activity of synaptosomes

These experiments examined effects of several ligands on the K⁺ p-nitrophenylphosphatase activity of the (Na⁺,K⁺)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K⁺-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K⁺ was approximately 75% less than that observed when K⁺ was included in the incubation medium. The response to the H⁺ concentrations was different: K⁺-independent activity showed a pH optimum around 6.5–7.0, while the K⁺-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K⁺-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K⁺-independent activity. Na⁺ did not affect K⁺-independent activity at all, while the same ligand concentration inhibited sharply the K⁺-dependent activity; this inhibition was not competitive with the substrate,p-nitrophenyl phosphate. K⁺-dependent activity was stimulated by Mg²⁺ with low affinity (millimolar range), and 3 mM Mg²⁺ produced a slight stimulation of the activity in absence of K⁺, which could be interpreted as Mg²⁺ occupying the K⁺ sites. Ca²⁺ had no appreciable effect on the activity in the absence of K⁺. However, in the presence of K⁺ a sharp inhibition was found with all Ca²⁺ concentrations studied. ATP (0.5 mM) did not affect the K⁺-independent activity, but this nucleotide behaved as a competitive inhibitor top-nitrophenylphosphate. Pi inhibited activity in the presence of K⁺, competively to the substrate, so it could be considered as the second product of the reaction sequence.Abbreviations used p-NPP p-nitrophenylphosphate - p-NPPase rho-nitrophenylphosphatase activity     

The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations

The Na+,K+-ATPase transforms the energy of ATP to the maintenance of steep electrochemical gradients for sodium and potassium across the plasma membrane. This activity is tissue specific, in particular due to variations in the expressions of the alpha subunit isoforms one through four. Several mutations in alpha2 and 3 have been identified that link the specific function of the Na+,K+-ATPase to the pathophysiology of neurological diseases such as rapid-onset dystonia parkinsonism and familial hemiplegic migraine type 2. We show a mapping of the isoform differences and the disease-related mutations on the recently determined crystal structure of the pig renal Na+,K+-ATPase and a structural comparison to Ca2+-ATPase. Furthermore, we present new experimental data that address the role of a stretch of three conserved arginines near the C-terminus of the alpha subunit (Arg1003-Arg1005).     

The carbonyl group of glutamic acid-795 is essential for gastric H+,K+-ATPase activity

To study the role of Glu795offresent in the fifth transmembrane domain of the alpha-subunit of gastric H+,K+-ATPase, several mutants were generated and expressed in Sf9 insect cells. The E795Q mutant had rather similar properties as the wild-type enzyme. The apparent affinity for K+ in both the ATPase reaction and the dephosphorylation of the phosphorylated intermediate was even slightly enhanced. This indicates that the carbonyl group of Glu795 is sufficient for enzymatic activity. This carbonyl group, however, has to be at a particular position with respect to the other liganding groups, since the E795D and E795N mutants showed a strongly reduced ATPase activity, a lowered apparent K+ affinity, and a decreased steady-state phosphorylation level. In the absence of a carbonyl residue at position 795, the K+ sensitivity was either strongly decreased (E795A) or completely absent (E795L). The mutant E795L, however, showed a SCH 28080 sensitive ATPase activity in the absence of K+, as well as an enhanced spontaneous dephosphorylation rate, that could not be further enhanced by K+, suggesting that this mutant mimicks the filled K+ binding pocket. The results indicate that the Glu795 residue is involved in K+-stimulated ATPase activity and K+-induced dephosphorylation of the phosphorylated intermediate. Glu795 might also be involved in H+ binding during the phosphorylation step, since the mutants E795N, E795D, and E795A showed a decrease in the phosphorylation rate as well as in the apparent ATP affinity in the phosphorylation reaction. This indicates that Glu795 is not only involved in K+ but might also play a role in H+ binding.     

Phe(475) and Glu(446) but not Ser(445) participate in ATP-binding to the alpha-subunit of Na(+)/K(+)-ATPase

The ATP-binding site of Na(+)/K(+)-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H(4) and H(5). Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.