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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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CPZ increases K+-independent Na+, K+-ATPase activity.A. Raw trace of the effects of treatment with 30 µM of CPZ on K+-independent Ip followed by the addition of 10 mM ouabain. B. Changes in steady state K+-independent Ip as a result of CPZ and CPZ plus ouabain, normalized to the K+-free control Ip. The data represent cells isolated from 6 individual animals [number of cells are shown inside bars] and are expressed as mean ±SEM (*significantly difference from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test.
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pone-0096909-g002: CPZ increases K+-independent Na+, K+-ATPase activity.A. Raw trace of the effects of treatment with 30 µM of CPZ on K+-independent Ip followed by the addition of 10 mM ouabain. B. Changes in steady state K+-independent Ip as a result of CPZ and CPZ plus ouabain, normalized to the K+-free control Ip. The data represent cells isolated from 6 individual animals [number of cells are shown inside bars] and are expressed as mean ±SEM (*significantly difference from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test.

Mentions: In order to determine whether the increase in K+-independent Ip (Fig. 1C) is ouabain-sensitive, stimulation of K+-independent Ip by CPZ was allowed to occur and then ouabain (10 mM) was added. Ouabain completely inhibited the CPZ-induced increase in K+-independent Ip (Fig. 2).


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)

CPZ increases K+-independent Na+, K+-ATPase activity.A. Raw trace of the effects of treatment with 30 µM of CPZ on K+-independent Ip followed by the addition of 10 mM ouabain. B. Changes in steady state K+-independent Ip as a result of CPZ and CPZ plus ouabain, normalized to the K+-free control Ip. The data represent cells isolated from 6 individual animals [number of cells are shown inside bars] and are expressed as mean ±SEM (*significantly difference from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096909-g002: CPZ increases K+-independent Na+, K+-ATPase activity.A. Raw trace of the effects of treatment with 30 µM of CPZ on K+-independent Ip followed by the addition of 10 mM ouabain. B. Changes in steady state K+-independent Ip as a result of CPZ and CPZ plus ouabain, normalized to the K+-free control Ip. The data represent cells isolated from 6 individual animals [number of cells are shown inside bars] and are expressed as mean ±SEM (*significantly difference from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test.
Mentions: In order to determine whether the increase in K+-independent Ip (Fig. 1C) is ouabain-sensitive, stimulation of K+-independent Ip by CPZ was allowed to occur and then ouabain (10 mM) was added. Ouabain completely inhibited the CPZ-induced increase in K+-independent Ip (Fig. 2).

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