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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 under Na+,Na+ exchange conditions.Oxonol VI fluorescence was measured as described in Materials and Methods. The working volume in the cuvette throughout the measurement was 2–2.1 ml. The cuvette contained 30 mM histidine, 30 mM NaCl, 2 mM MgCl2, and 20–30 µl proteoliposomes, and the experiment was started with the addition of 75 µM ATP. After reaching steady state, 20 µM CPZ dissolved in DMSO was added. A. Shown is the effect of addition of ATP and CPZ in the absence (black) or in the presence (red) of 25 µM ADP. The numbers indicate the percentage decrease in maximum fluorescence occurred by pretreatment with 25 µM ADP. Representative of four independent experiments is shown. B. The fluorescence response was measured as in panel A, but CPZ was added first, followed by the addition of ATP, as indicated by the arrows.
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pone-0096909-g009: Effect of CPZ on oxonol VI fluorescence under Na+,Na+ exchange conditions.Oxonol VI fluorescence was measured as described in Materials and Methods. The working volume in the cuvette throughout the measurement was 2–2.1 ml. The cuvette contained 30 mM histidine, 30 mM NaCl, 2 mM MgCl2, and 20–30 µl proteoliposomes, and the experiment was started with the addition of 75 µM ATP. After reaching steady state, 20 µM CPZ dissolved in DMSO was added. A. Shown is the effect of addition of ATP and CPZ in the absence (black) or in the presence (red) of 25 µM ADP. The numbers indicate the percentage decrease in maximum fluorescence occurred by pretreatment with 25 µM ADP. Representative of four independent experiments is shown. B. The fluorescence response was measured as in panel A, but CPZ was added first, followed by the addition of ATP, as indicated by the arrows.

Mentions: Investigation of changes in the fluorescence of oxonol VI was used to gain information on the effect of CPZ on Na+ translocation via the pump. ATP-dependent transport of positive charge from the medium into the liposomal lumen, establishing an inside positive membrane potential, is detected by an increase in oxonol VI fluorescence [39]. As seen from Fig. 9A, addition of ATP in the presence of 30 mM Na+ on both sides of the membrane (3Na+:2Na+ exchange conditions) resulted in an expected increase in fluorescence (Fig 9A, black), indicative of electrogenic Na+ exchange with net influx of Na+ into the liposomes through reconstituted inside-out Na+,K+-ATPase molecules (cellular efflux). The fluorescence level reaches a plateau as the potential difference attains a level high enough to impair Na+ release from the E2P(Na+) form. Addition of CPZ at the plateau phase resulted in ∼100% increase in fluorescence to a new plateau. The CPZ effect was pump-mediated since CPZ produced no effect on oxonol VI fluorescence in the absence of the protein (data not shown). Furthermore, addition of CPZ before ATP did not produce any change in fluorescence (Fig. 9B); an increase in fluorescence is only observed following the addition of ATP.


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 under Na+,Na+ exchange conditions.Oxonol VI fluorescence was measured as described in Materials and Methods. The working volume in the cuvette throughout the measurement was 2–2.1 ml. The cuvette contained 30 mM histidine, 30 mM NaCl, 2 mM MgCl2, and 20–30 µl proteoliposomes, and the experiment was started with the addition of 75 µM ATP. After reaching steady state, 20 µM CPZ dissolved in DMSO was added. A. Shown is the effect of addition of ATP and CPZ in the absence (black) or in the presence (red) of 25 µM ADP. The numbers indicate the percentage decrease in maximum fluorescence occurred by pretreatment with 25 µM ADP. Representative of four independent experiments is shown. B. The fluorescence response was measured as in panel A, but CPZ was added first, followed by the addition of ATP, as indicated by the arrows.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096909-g009: Effect of CPZ on oxonol VI fluorescence under Na+,Na+ exchange conditions.Oxonol VI fluorescence was measured as described in Materials and Methods. The working volume in the cuvette throughout the measurement was 2–2.1 ml. The cuvette contained 30 mM histidine, 30 mM NaCl, 2 mM MgCl2, and 20–30 µl proteoliposomes, and the experiment was started with the addition of 75 µM ATP. After reaching steady state, 20 µM CPZ dissolved in DMSO was added. A. Shown is the effect of addition of ATP and CPZ in the absence (black) or in the presence (red) of 25 µM ADP. The numbers indicate the percentage decrease in maximum fluorescence occurred by pretreatment with 25 µM ADP. Representative of four independent experiments is shown. B. The fluorescence response was measured as in panel A, but CPZ was added first, followed by the addition of ATP, as indicated by the arrows.
Mentions: Investigation of changes in the fluorescence of oxonol VI was used to gain information on the effect of CPZ on Na+ translocation via the pump. ATP-dependent transport of positive charge from the medium into the liposomal lumen, establishing an inside positive membrane potential, is detected by an increase in oxonol VI fluorescence [39]. As seen from Fig. 9A, addition of ATP in the presence of 30 mM Na+ on both sides of the membrane (3Na+:2Na+ exchange conditions) resulted in an expected increase in fluorescence (Fig 9A, black), indicative of electrogenic Na+ exchange with net influx of Na+ into the liposomes through reconstituted inside-out Na+,K+-ATPase molecules (cellular efflux). The fluorescence level reaches a plateau as the potential difference attains a level high enough to impair Na+ release from the E2P(Na+) form. Addition of CPZ at the plateau phase resulted in ∼100% increase in fluorescence to a new plateau. The CPZ effect was pump-mediated since CPZ produced no effect on oxonol VI fluorescence in the absence of the protein (data not shown). Furthermore, addition of CPZ before ATP did not produce any change in fluorescence (Fig. 9B); an increase in fluorescence is only observed following the addition of ATP.

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