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Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump.

ABSTRACT: The Na/K pump actively exports 3 Na(+) in exchange for 2 K(+) across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the alpha-subunit diminishes internal Na(+) (Na(i)(+)) affinity approximately 25-fold [Morth et al. (2007) Nature 450:1043-1049]. Because external Na(+) (Na(o)(+)) binding is voltage-dependent, we studied the reactions involving this process by using two-electrode and inside-out patch voltage clamp in normal and truncated (DeltaKESYY) Xenopus-alpha1 pumps expressed in oocytes. We observed that DeltaKESYY (i) decreased both Na(o)(+) and Na(i)(+) apparent affinities in the absence of K(o)(+), and (ii) did not affect apparent Na(o)(+) affinity at high K(o)(+). These results support a model of strict sequential external release of Na(+) ions, where the Na(+)-exclusive site releases Na(+) before the sites shared with K(+) and the DeltaKESYY deletion only reduces Na(o)(+) affinity at the shared sites. Moreover, at nonsaturating K(o)(+), DeltaKESYY induced an inward flow of Na(+) through Na/K pumps at negative potentials. Guanidinium(+) can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidinium(o)(+) can also traverse normal Na/K pumps in the absence of both Na(o)(+) and K(o)(+) and can also inhibit Na/K pump currents in a Na(+)-like voltage-dependent manner, we conclude that the normal pathway transited by the first externally released Na(+) is large enough to accommodate guanidinium(+).

SUBMITTER: Yaragatupalli S

PROVIDER: S-EPMC2741281 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump.

Yaragatupalli Siddhartha S Olivera J Fernando JF Gatto Craig C Artigas Pablo P

Proceedings of the National Academy of Sciences of the United States of America 20090824 36

The Na/K pump actively exports 3 Na(+) in exchange for 2 K(+) across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the alpha-subunit diminishes internal Na(+) (Na(i)(+)) affinity approximately 25-fold [Morth et al. (2007) Nature 450:1043-1049]. Because external Na(+) (Na(o ...[more]

PMID: 19706387

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Project description:Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ?-75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ?+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

Project description:In this study the possible role of Na+ influx, arachidonate mediators and alpha-subunit phosphorylation in the stimulatory response of hepatic Na+/K(+)-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive 86Rb+ uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4( +/- 1.3) x 10(-10) M. Analysis of the K(+)-dependence of this response indicates an effect on the apparent Vmax. for K+ with no significant change in the apparent kappa 0.5. Unlike monensin, glucagon stimulation of Na+/K(+)-ATPase-mediated transport activity was not associated with an increase in 22Na+ influx. This indicates that the stimulation of Na+/K(+)-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive 86Rb+ uptake was significantly affected by supramaximal concentrations of cyclo-oxygenase, lipoxygenase, cytochrome p-450 or phospholipase A2 inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive 86Rb uptake, hepatocytes were metabolically radiolabelled with [32P]P(i), Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that comigrates with Na+/K(+)-ATPase alpha-subunit immunoreactivity in two-dimensional gel electrophoresis. The alpha-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K(+)-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]P(i), the immunoprecipitated alpha-subunit contained 32P. Glucagon increased the incorporation of 32P into the immunoprecipitated subunit by 197 +/- 21% (n = 6). Similar results were observed with a rabbit anti-peptide serum ('anti-LEAVE' serum) prepared against an amino acid sequence in the alpha-subunit. The EC50 for glucagon-stimulated phosphorylation of the alpha-subunit (approximately 1 x 10(-10) M) was very close to that for glucagon stimulation of ouabain-sensitive 86Rb+ uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K(+)-ATPase-mediated transport activity is not secondary to increases in Na+ influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na(+)-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the alpha-subunit.

Project description:The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.

Dataset Information

Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump.

Publications

Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump.

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