Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of CPZ on pNPP hydrolysis by Na+,K+-ATPase.pNPPase activity was measured in the presence of histidine buffer (with the pH adjusted with Tris/HCl), 10 mM MgCl2, 10 mM Na2+-pNPP, 100 mM NaCl (A) or 100 mM KCl (B) in the presence of the indicated CPZ concentrations and in the absence of ATP. Released pNP was measured in a spectrophotometer at 410 nm as described under Materials and Methods. Data are expressed as µmol hydrolyzed pNPP · h−1 · mg−1 (at 24°C). The black circles depict the effect of linoleic acid on pNPPase activity in the presence of 100 mM Na+ at concentrations identical to those of CPZ. Note that the residual activity seen in panel B at high CPZ concentrations is likely due to the presence of 20 mM Na+ added with pNPP.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4016139&req=5

pone-0096909-g005: Effect of CPZ on pNPP hydrolysis by Na+,K+-ATPase.pNPPase activity was measured in the presence of histidine buffer (with the pH adjusted with Tris/HCl), 10 mM MgCl2, 10 mM Na2+-pNPP, 100 mM NaCl (A) or 100 mM KCl (B) in the presence of the indicated CPZ concentrations and in the absence of ATP. Released pNP was measured in a spectrophotometer at 410 nm as described under Materials and Methods. Data are expressed as µmol hydrolyzed pNPP · h−1 · mg−1 (at 24°C). The black circles depict the effect of linoleic acid on pNPPase activity in the presence of 100 mM Na+ at concentrations identical to those of CPZ. Note that the residual activity seen in panel B at high CPZ concentrations is likely due to the presence of 20 mM Na+ added with pNPP.

Mentions: Na+,K+-ATPase hydrolyses pNPP in a partial reaction involving an E2 form, and is detected following incubation of the enzyme in the presence of K+ (E2K+ form), or in the presence of Na+ and MgATP (E2P form). pNPP hydrolysis is inhibited by ligands that stabilize the E1 conformation and is therefore not observed when Na+ substitutes K+. We have now found that CPZ stimulates pNPP hydrolysis in the presence of 100 mM Na+ and in the absence of K+ (Fig. 5A). The maximum level of stimulation was independent of pH although the apparent affinity of inhibition by CPZ decreased at basic pH. Linoleic acid, a free fatty acid that stabilizes the E2 conformation [32], does not produce a similar effect (Fig. 5A, closed circles). On the other hand, K+-dependent pNPPase is inhibited by CPZ at all pH values tested, but the inhibition was found to be strongly dependent on pH (Fig. 5B); the apparent affinity of inhibition by CPZ was much higher at basic pH compared to acidic and neutral pH.


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 pNPP hydrolysis by Na+,K+-ATPase.pNPPase activity was measured in the presence of histidine buffer (with the pH adjusted with Tris/HCl), 10 mM MgCl2, 10 mM Na2+-pNPP, 100 mM NaCl (A) or 100 mM KCl (B) in the presence of the indicated CPZ concentrations and in the absence of ATP. Released pNP was measured in a spectrophotometer at 410 nm as described under Materials and Methods. Data are expressed as µmol hydrolyzed pNPP · h−1 · mg−1 (at 24°C). The black circles depict the effect of linoleic acid on pNPPase activity in the presence of 100 mM Na+ at concentrations identical to those of CPZ. Note that the residual activity seen in panel B at high CPZ concentrations is likely due to the presence of 20 mM Na+ added with pNPP.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096909-g005: Effect of CPZ on pNPP hydrolysis by Na+,K+-ATPase.pNPPase activity was measured in the presence of histidine buffer (with the pH adjusted with Tris/HCl), 10 mM MgCl2, 10 mM Na2+-pNPP, 100 mM NaCl (A) or 100 mM KCl (B) in the presence of the indicated CPZ concentrations and in the absence of ATP. Released pNP was measured in a spectrophotometer at 410 nm as described under Materials and Methods. Data are expressed as µmol hydrolyzed pNPP · h−1 · mg−1 (at 24°C). The black circles depict the effect of linoleic acid on pNPPase activity in the presence of 100 mM Na+ at concentrations identical to those of CPZ. Note that the residual activity seen in panel B at high CPZ concentrations is likely due to the presence of 20 mM Na+ added with pNPP.
Mentions: Na+,K+-ATPase hydrolyses pNPP in a partial reaction involving an E2 form, and is detected following incubation of the enzyme in the presence of K+ (E2K+ form), or in the presence of Na+ and MgATP (E2P form). pNPP hydrolysis is inhibited by ligands that stabilize the E1 conformation and is therefore not observed when Na+ substitutes K+. We have now found that CPZ stimulates pNPP hydrolysis in the presence of 100 mM Na+ and in the absence of K+ (Fig. 5A). The maximum level of stimulation was independent of pH although the apparent affinity of inhibition by CPZ decreased at basic pH. Linoleic acid, a free fatty acid that stabilizes the E2 conformation [32], does not produce a similar effect (Fig. 5A, closed circles). On the other hand, K+-dependent pNPPase is inhibited by CPZ at all pH values tested, but the inhibition was found to be strongly dependent on pH (Fig. 5B); the apparent affinity of inhibition by CPZ was much higher at basic pH compared to acidic and neutral pH.

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