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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 decreases K+-dependent Na+, K+-ATPase transport activity.A. Raw trace of the effects of 30 µM of CPZ on Na+, K+-ATPase Ip in adult rat cardiac myocytes. The horizontal bold lines indicate the addition and wash-off of 5 mM KCl or the addition of CPZ, as indicated. B. Steady state current obtained following 5 and 10 min incubation with CPZ, compared to controls not treated with CPZ. C. Changes in K+-independent Ip following the addition of CPZ. D. The CPZ-induced changes of Na+, K+-ATPase Ip in adult rat cardiac myocytes are reversible. The data represent cells isolated from at least 6 individual animals, (number of cells shown inside bars; 1 min time point not shown) and are expressed as mean ±SEM (*significantly different from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test in panel B and by unpaired t-test in panel C.
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pone-0096909-g001: CPZ decreases K+-dependent Na+, K+-ATPase transport activity.A. Raw trace of the effects of 30 µM of CPZ on Na+, K+-ATPase Ip in adult rat cardiac myocytes. The horizontal bold lines indicate the addition and wash-off of 5 mM KCl or the addition of CPZ, as indicated. B. Steady state current obtained following 5 and 10 min incubation with CPZ, compared to controls not treated with CPZ. C. Changes in K+-independent Ip following the addition of CPZ. D. The CPZ-induced changes of Na+, K+-ATPase Ip in adult rat cardiac myocytes are reversible. The data represent cells isolated from at least 6 individual animals, (number of cells shown inside bars; 1 min time point not shown) and are expressed as mean ±SEM (*significantly different from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test in panel B and by unpaired t-test in panel C.

Mentions: In order to investigate the functional effects of CPZ on Na+,K+-ATPase activity in a more integrated system, pump current (Ip) was measured in single freshly isolated adult rat ventricular cardiomyocytes using a whole-cell ruptured patch clamp technique [22], [23]. Ip was defined as current inhibited by removal of extracellular K+ (K+-dependent current). Ip was measured in Tyrode solution followed by treatment with either DMSO vehicle control or 30 µM CPZ. Perfusion of the cardiomyocytes with solution containing CPZ decreased K+-dependent current, as shown in Fig. 1A. Five min of perfusion with CPZ decreased Ip by 52% (Ip decreased from 2.246 ± 0.171 to 1.172±0.1607 pA/pF; p<0.05, n = 11), whereas, 10 min of perfusion with CPZ resulted in 85% inhibition (Ip = 0.3250±0.2136 pA/pF; p<0.05, n = 7), as shown in Fig. 1B.


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 decreases K+-dependent Na+, K+-ATPase transport activity.A. Raw trace of the effects of 30 µM of CPZ on Na+, K+-ATPase Ip in adult rat cardiac myocytes. The horizontal bold lines indicate the addition and wash-off of 5 mM KCl or the addition of CPZ, as indicated. B. Steady state current obtained following 5 and 10 min incubation with CPZ, compared to controls not treated with CPZ. C. Changes in K+-independent Ip following the addition of CPZ. D. The CPZ-induced changes of Na+, K+-ATPase Ip in adult rat cardiac myocytes are reversible. The data represent cells isolated from at least 6 individual animals, (number of cells shown inside bars; 1 min time point not shown) and are expressed as mean ±SEM (*significantly different from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test in panel B and by unpaired t-test in panel C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096909-g001: CPZ decreases K+-dependent Na+, K+-ATPase transport activity.A. Raw trace of the effects of 30 µM of CPZ on Na+, K+-ATPase Ip in adult rat cardiac myocytes. The horizontal bold lines indicate the addition and wash-off of 5 mM KCl or the addition of CPZ, as indicated. B. Steady state current obtained following 5 and 10 min incubation with CPZ, compared to controls not treated with CPZ. C. Changes in K+-independent Ip following the addition of CPZ. D. The CPZ-induced changes of Na+, K+-ATPase Ip in adult rat cardiac myocytes are reversible. The data represent cells isolated from at least 6 individual animals, (number of cells shown inside bars; 1 min time point not shown) and are expressed as mean ±SEM (*significantly different from control, P<0.05). Differences between experimental groups were tested by one-way ANOVA followed by a Bonferroni post-hoc test in panel B and by unpaired t-test in panel C.
Mentions: In order to investigate the functional effects of CPZ on Na+,K+-ATPase activity in a more integrated system, pump current (Ip) was measured in single freshly isolated adult rat ventricular cardiomyocytes using a whole-cell ruptured patch clamp technique [22], [23]. Ip was defined as current inhibited by removal of extracellular K+ (K+-dependent current). Ip was measured in Tyrode solution followed by treatment with either DMSO vehicle control or 30 µM CPZ. Perfusion of the cardiomyocytes with solution containing CPZ decreased K+-dependent current, as shown in Fig. 1A. Five min of perfusion with CPZ decreased Ip by 52% (Ip decreased from 2.246 ± 0.171 to 1.172±0.1607 pA/pF; p<0.05, n = 11), whereas, 10 min of perfusion with CPZ resulted in 85% inhibition (Ip = 0.3250±0.2136 pA/pF; p<0.05, n = 7), as shown in Fig. 1B.

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