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Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

Mahmmoud YA, Shattock M, Cornelius F, Pavlovic D - PLoS ONE (2014)

Bottom Line: Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity.Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green.This effect of guanidinium was amplified by treatment with CPZ.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark.

ABSTRACT
Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

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Effect of CPZ on oxonol VI fluorescence in the presence of 10 folds ion gradient across liposomal membrane.Liposomes were equilibrated with either 300(A) or 300 mM NaCl (B). The medium contained 30 mM histidine, 2 mM MgCl2 and 20 µl proteoliposomes. CPZ was added first, followed by 75 µl Tris-ATP, as indicated by the arrows.
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pone-0096909-g011: Effect of CPZ on oxonol VI fluorescence in the presence of 10 folds ion gradient across liposomal membrane.Liposomes were equilibrated with either 300(A) or 300 mM NaCl (B). The medium contained 30 mM histidine, 2 mM MgCl2 and 20 µl proteoliposomes. CPZ was added first, followed by 75 µl Tris-ATP, as indicated by the arrows.

Mentions: A surprising effect was observed in experiments where the effect of CPZ was studied in the presence of a high extravesicular concentration (300 mM) of either K+ or Na+. In the presence of a K+ gradient, a substantial increase in fluorescence occurred following addition of CPZ in the absence of ATP (Fig. 11A), and subsequent addition of ATP produced only a very small increase in fluorescence. In contrast, in the presence of 300 mM Na+ in the extravesicular medium, CPZ had no effect on fluorescence in the absence of ATP, whereas subsequent addition of ATP increased the fluorescence in a manner similar to that found in Fig. 9A (Fig. 11B). This CPZ effect was not influenced by the addition of digitoxigenin during a pre-incubation period of 5 min (data not shown).


Inhibition of K+ transport through Na+, K+-ATPase by capsazepine: role of membrane span 10 of the α-subunit in the modulation of ion gating.

Mahmmoud YA, Shattock M, Cornelius F, Pavlovic D - PLoS ONE (2014)

Effect of CPZ on oxonol VI fluorescence in the presence of 10 folds ion gradient across liposomal membrane.Liposomes were equilibrated with either 300(A) or 300 mM NaCl (B). The medium contained 30 mM histidine, 2 mM MgCl2 and 20 µl proteoliposomes. CPZ was added first, followed by 75 µl Tris-ATP, as indicated by the arrows.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016139&req=5

pone-0096909-g011: Effect of CPZ on oxonol VI fluorescence in the presence of 10 folds ion gradient across liposomal membrane.Liposomes were equilibrated with either 300(A) or 300 mM NaCl (B). The medium contained 30 mM histidine, 2 mM MgCl2 and 20 µl proteoliposomes. CPZ was added first, followed by 75 µl Tris-ATP, as indicated by the arrows.
Mentions: A surprising effect was observed in experiments where the effect of CPZ was studied in the presence of a high extravesicular concentration (300 mM) of either K+ or Na+. In the presence of a K+ gradient, a substantial increase in fluorescence occurred following addition of CPZ in the absence of ATP (Fig. 11A), and subsequent addition of ATP produced only a very small increase in fluorescence. In contrast, in the presence of 300 mM Na+ in the extravesicular medium, CPZ had no effect on fluorescence in the absence of ATP, whereas subsequent addition of ATP increased the fluorescence in a manner similar to that found in Fig. 9A (Fig. 11B). This CPZ effect was not influenced by the addition of digitoxigenin during a pre-incubation period of 5 min (data not shown).

Bottom Line: Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity.Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green.This effect of guanidinium was amplified by treatment with CPZ.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine, University of Aarhus, DK-8000 Aarhus C, Denmark.

ABSTRACT
Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4-.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

Show MeSH
Related in: MedlinePlus