Evidence for altered ion transport in Saccharomyces cerevisiae overexpressing human MDR 1 protein

Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11168] we presented evidence for a novel Na+- and Cl--dependent H+ transport process in LR73/hu MDR 1 CHO transfectants that likely explains pHi, volume, and membrane potential changes in eukaryotic cells overexpressing the hu MDR 1 protein. To further explore this process, we have overexpressed human MDR 1 protein in yeast strain 9.3 following a combination of approaches used previously [Kuchler, K., and Thorner, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2302-2306; Ruetz, S., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11588-11592]. Thus, a truncated hu MDR 1 cDNA was cloned behind a tandem array of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters to create the yeast expression vector pFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasma membranes confirmed the overexpression of full length, functional, and properly localized hu MDR 1 protein in independently isolated 9.3/pFF1 colonies. Interestingly, relative valinomycin resistance and growth of the 9.3/hu MDR 1 strains are found to strongly depend on the ionic composition of the growth medium. Atomic absorption reveals significant differences in intracellular K+ for 9.3/hu MDR 1 versus control yeast. Transport assays using [3H]tetraphenylphosphonium ([3H]TPP+) reveal perturbations in membrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pHex. ATPase activity of purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectants constructed previously [Hoffman, M. M., et al. (1996) J. Gen. Physiol. 108, 295-313] was compared. MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeast H+ ATPase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and control strains were analyzed for formation of H+ gradients +/- verapamil. Similar pharmacologic profiles are found for verapamil stimulation of MDR 1 ATPase activity and H+ pumping in 9.3/hu MDR 1 ISOV. In sum, these experiments strongly support the notion that hu MDR 1 catalyzes H+ transport in some fashion and lowers membrane potential in yeast when K+ contributes strongly to that potential. In the accompanying paper [Santai, C. T., Fritz, F., and Roepe, P. D. (1999) Biochemistry 38, XXXX-XXXX] the effects of ion gradients on H+ transport by hu MDR 1 are examined.     

Are the states that occlude rubidium obligatory intermediates of the Na(+)/K(+)-ATPase reaction?

In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.     

Ionophore-mediated coupling between ion fluxes and amino acid absorption in mouse ascites-tumour cells. Restoration of the physiological gradients of methionine by valinomycin in the absence of adenosine triphosphate

1. Preparations of mouse ascites-tumour cells depleted of ATP and Na(+) ions accumulated l-methionine, in the presence of cyanide and deoxyglucose, from a 1mm solution containing 80mequiv. of Na(+)/l and about 5mequiv. of K(+)/l. Valinomycin increased, from about 4 to 16, the maximum value of the ratio of the cellular to extracellular concentrations of methionine formed under these conditions without markedly affecting the distributions of Na(+) and of K(+). Similar observations were made with 2-aminoisobutyrate, glycine and l-leucine. Increasing the extracellular concentration of K(+) progressively decreased the accumulation of methionine in the presence of valinomycin. Over the physiological range of ionic gradients, the system behaved as though the absorption of methionine with Na(+) was closely coupled to the electrogenic efflux of K(+) through the ionophore. The process was insensitive to ouabain and so the sodium pump was probably not involved. 2. The amount of methionine accumulated during energy metabolism was similar to the optimal accumulation in the presence of valinomycin when ATP was lacking. It was also similarly affected by increasing the methionine concentration. 3. A mixture of nigericin and tetrachlorosalicylanilide mimicked the action of valinomycin. The anilide derivative inhibited the absorption of 2-aminoisobutyrate in the presence of valinomycin, but not in its absence. 4. Gramicidin inhibited methionine absorption and caused the preparations to absorb Na(+) and lose K(+). 5. The observations appear to verify the principle underlying the gradient hypothesis by showing that the tumour cells can efficiently couple the electrochemical gradient of Na(+) to the amino acid gradient.     

The arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes.

In saline environments, plants accumulate Na(+) in vacuoles through the activity of tonoplast Na(+)/H(+) antiporters. The first gene for a putative plant vacuolar Na(+)/H(+) antiporter, AtNHX1, was isolated from Arabidopsis and shown to increase plant tolerance to NaCl. However, AtNHX1 mRNA was up-regulated by Na(+) or K(+) salts in plants and substituted for the homologous protein of yeast to restore tolerance to several toxic cations. To study the ion selectivity of the AtNHX1 protein, we have purified a histidine-tagged version of the protein from yeast microsomes by Ni(2+) affinity chromatography, reconstituted the protein into lipid vesicles, and measured cation-dependent H(+) exchange with the fluorescent pH indicator pyranine. The protein catalyzed Na(+) and K(+) transport with similar affinity in the presence of a pH gradient. Li(+) and Cs(+) ions were also transported with lower affinity. Ion exchange by AtNHX1 was inhibited 70% by the amiloride analog ethylisopropyl-amiloride. Our data indicate a role for intracellular antiporters in organelle pH control and osmoregulation.     

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,β,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the β-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular β,β-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.     

大豆液泡膜H~+-ATPase的纯化和重组

通过不连续蔗糖密度梯度离心得到的液泡膜微囊 ,先由胆酸钠和 OG分步破膜抽提、经阴离子交换柱 ( Q- Sepharose)层析分离 .纯化后的酶含 V型 H+ - ATPase的主要亚基 ,与大豆磷脂重组 ,获得了有较高泵活性的脂酶体 .脂酶体的质子泵活性受 Valinomycin激活 ,说明它是致电性的 ,受NO-3 ,DCCD以及特异性的 V型 ATPase抑制剂 Bafilomycin的抑制 .脂酶体的泵活性不受 F型和P型 ATPase抑制剂抑制 ,表明质子转运是由 V型 H+ - ATPase引起的 .     

Proton diet for the sodium pump

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

Activation of P2Y2 purinoceptor inhibits the activity of the Na+/K+-ATPase in HeLa cells

The role of ATP on regulation of the Na(+)/K(+)-ATPase activity in the human cancerous HeLa cells was investigated. HeLa cells stimulated with increasing ATP concentrations showed a dose-dependent inhibition of the Na(+)/K(+)-ATPase activity. These effects were also obtained by UTP. ATP and UTP provoked a rise in intracellular calcium concentration ([Ca(2+)](i)) persisting for at least 4 min. The inhibitor of phospholipase C, U73122, blocked the elevation of [Ca(2+)](i) provoked by ATP/UTP. The expression of mRNA for P2Y2 and P2Y6 receptors was demonstrated by RT-PCR. ATP/UTP activated PKC-alpha, -betaI and -epsilon isoforms, but not PKC-delta and -zeta. The inhibition of the Na(+)/K(+)-ATPase activity by ATP/UTP was blocked by G?6976, a specific inhibitor of the calcium-dependent PKCs. In conclusion, our results suggest that ATP/UTP modulate Na(+)/K(+)-ATPase activity in HeLa cells through the P2Y2 purinoceptor via calcium mobilisation and activation of calcium-dependent PKCs.     

Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation

Short-term exposure of coho salmon smolts (Oncorhynchus kisutch) to a gradual increase in salinity over 2 d (0 per thousand -32 per thousand ) resulted in a decrease in proton pump abundance, detected as changes in immunoreactivity with a polyclonal antibody against subunit A of bovine brain vacuolar H(+)-ATPase. N-ethylmaleimide (NEM)-sensitive H(+)-ATPase activities in gill homogenates remained unchanged over 8 d to coincide with a 3.5-fold increase in Na(+)/K(+)-ATPase activities. A transient increase in plasma [Na(+)] and [Cl(-)] levels over the 8-d period was preceded by a 10-fold increase in plasma cortisol levels, which peaked after 12 h. Long-term (1 mo) acclimation to seawater resulted in the loss of apical immunoreactivity for vH(+)-ATPase and band 3-like anion exchanger in the mitochondria-rich cells identified by high levels of Na(+)/K(+)-ATPase immunoreactivity. The polyclonal antibody Ab597 recognized a Na(+)/H(+) exchanger (NHE-2)-like protein in what appears to be an accessory cell (AC) type. Populations of these ACs were found associated with Na(+)/K(+)-ATPase rich chloride cells in both freshwater- and seawater-acclimated animals.     

Chemiosmotic ATP synthesis in photosynthetically inactive chromoplasts from Narcissus pseudonarcissus L. linked to a redox pathway potentially also involved in carotene desaturation

Mature chromoplasts from daffodil (Narcissus pseudonarcissus) flowers, although devoid of thylakoid structures, contain immunologically detectable alpha-subunits of ATP-synthase (H(+)-transporting ATP phosphohydrolase; EC 3.6.3.14). To show the presence of the entire functional protein complex, chromoplast membrane proteins were solubilized and reconstituted in phosphatidylcholine liposomes. The membranes were energized by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential, and the initial rate of ATP synthesis was measured with a luciferin/luciferase assay. In addition, by demonstrating NADPH-dependent ATP synthesis, we show that an NAD(P)H-dependent respiratory redox pathway in chromoplasts, previously identified as an important constituent of the carotene desaturation system, proceeds concomitant with membrane energization.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Effects of ion gradients on H+ transport mediated by human MDR 1 protein

In the previous paper we presented a variety of data consistent with significant perturbations in 9.3 yeast plasma membrane ion transport upon overexpression of the hu MDR 1 protein. Thus, in this paper, we compare formation of DeltapH for inside-out yeast plasma membrane vesicles (ISOV) prepared from control 9.3/pVT versus 9.3/hu MDR 1 yeast. Since MDR 1 ATPase activity has a broader, more alkaline pH profile relative to endogenous yeast H+ ATPase activity, we analyzed H+ pumping at pH >/= 8.0 in detail in order to selectively amplify hu MDR 1 contributions to H+ movement over those of the endogenous yeast H+ ATPase. We observed: (1) imposition of a Cl- gradient oriented outside to in enhances acidification for 9.3/pVT ISOV (as expected), but decreases acidification for 9.3/hu MDR 1 ISOV; (2) imposition of a Cl- gradient oriented inside to out decreases acidification for 9.3/pVT ISOV (as expected) but enhances acidification for 9.3/hu MDR 1 ISOV; (3) a Na+ gradient oriented in the same direction as the Cl- gradient amplifies the effects due to hu MDR 1 when both gradients are oriented inside to out, but not outside to in. The data are most easily explained by interesting Na+, Cl-, and ATP-dependent H+ transport mediated by hu MDR 1 protein as previously suggested [Hoffman and Roepe (1997) Biochemistry 36, 11153-11168]. These data may help to resolve a variety of conflicting reports in the literature regarding ion transport mediated by hu MDR 1 and have implications for the physiology of a number of polarized epithelia in which hu MDR 1 is endogenously expressed.     

Functional reconstitution of influenza A M2(22-62)

Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K(+)] gradient-induced membrane potential and [H(+)] gradient), M2(22-62) showed a transport rate of 78 H(+)/tetramer-s (pH(o) 6.0, pH(i) 8.0, nominal V(m)=-114 mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05-1.0% (peptide wt.% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K(+) flux of 0.1K(+)/tetramer-s, and also demonstrated that the K(+) permeability, relative to H(+), was 2.8 × 10(-6). Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H(+) selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pK(a) of 6.     

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.     

Cation specificity of osmosensing by the betaine carrier BetP of Corynebacterium glutamicum

The Na(+)/betaine carrier BetP from Corynebacterium glutamicum was purified and reconstituted in Escherichia coli phospholipid liposomes and its osmosensory properties were studied with respect to the cation specificity of osmotic activation. To dissect the influence of the co-substrate Na(+) on the energetics of uptake from its possible role as a putative trigger of osmolality-dependent BetP activation, the internal Na(+) concentration was varied without changing DeltapNa(+). Studying betaine uptake at increasing luminal Na(+) or K(+) revealed that BetP activity was triggered by Na(+) only to a negligible extent compared to activation by K(+). We conclude that activation of BetP in proteoliposomes depends solely on K(+), both in mechanistic and in physiological terms.     

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.     

Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes.

In a previous work, we have shown that overexpression of the Na(+)/K(+)/Cl(-) cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c-NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP-ERK). Furthermore, the level of DP-ERK was reduced by 50-80% following the addition of bumetanide, a specific inhibitor of the Na(+)/K(+)/Cl(-) cotransporter, in quiescent as well as in proliferating cultures of the Balb/c-NKCC1 clone. In order to explore further the role of the Na(+)/K(+)/Cl(-) cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP-ERK level in immortalized non-transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50-60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K(+) influx mediated by the Na(+)/K(+)/Cl(-) cotransporter in these cells. To analyze whether the Na(+)/K(+)/Cl(-) cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na(+)/K(+)/Cl(-) cotransporter controls the ERK/MAPK (mitogen-activated protein kinase) signal transduction pathway, support our hypothesis that Na(+) and K(+) influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na(+)/K(+)/Cl(-) cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery.     

Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.     

Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H(+)-ATPase AHA2 of Arabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell. To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca(2+)-, Na(+)/K(+)-, H(+)/K(+)-, and H(+)-ATPases and which coordinates Ca(2+) ions in the SERCA1 Ca(2+)-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) --> Asn mutant, and provide evidence that Asp(684) in the plasma membrane H(+)-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H(+)-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed. The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E(2) conformation. During catalysis the Asp(684) --> Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E(1) conformation and is unable to proceed through the E(1)P-E(2)P transition.     

Sodium flux ratio in Na/K pump-channels opened by palytoxin

Palytoxin binds to Na(+)/K(+) pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and (22)Na(+) efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound "pump-channels" in outside-out patches was approximately 7 pS in symmetrical 500 mM [Na(+)], comparable to findings in other cells. In these high-[Na(+)], K(+)-free solutions, with 5 mM cytoplasmic [ATP], the K(0.5) for palytoxin action was approximately 70 pM. The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+). The reversal potential of palytoxin-elicited current under biionic conditions, with the same concentration of a different permeant cation on each side of the membrane, was independent of the concentration of those ions over the range 55-550 mM. In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28). These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.