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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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Proteinase K cleavage.Pig kidney membranes were incubated in 25/or 1 mM ADP, as indicated. The samples were treated with PK (PK/protein ratio ∼1∶50) and the mixture incubated for 40 min at 24°C, and terminated with SDS sample buffer acidified with TCA. Proteolytic products were separated using SDS-PAGE and protein fragments on the gel were transferred to PVDF membranes and visualized by Western blotting using NKA1012-1016 antibody. The labels to the left indicate the apparent molecular weights of the fragments, determined by the use of Precision plus protein standards from Bio-Rad, Cat#161-0363.
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pone-0096909-g006: Proteinase K cleavage.Pig kidney membranes were incubated in 25/or 1 mM ADP, as indicated. The samples were treated with PK (PK/protein ratio ∼1∶50) and the mixture incubated for 40 min at 24°C, and terminated with SDS sample buffer acidified with TCA. Proteolytic products were separated using SDS-PAGE and protein fragments on the gel were transferred to PVDF membranes and visualized by Western blotting using NKA1012-1016 antibody. The labels to the left indicate the apparent molecular weights of the fragments, determined by the use of Precision plus protein standards from Bio-Rad, Cat#161-0363.

Mentions: Cleavage with PK of the cytoplasmic domains of the α-subunit was used as a tool to investigate the effect of CPZ on the conformation of pig kidney Na+,K+-ATPase. In the presence of occluded ions, a specific cleavage pattern is obtained as proteolytic sites at distinct locations are protected and others are exposed. In the absence of occluded ions, this protection is absent and all of the cytoplasmic domains of the α-subunit are completely cleaved. To this end, membrane-bound enzyme was incubated in the presence of different ion combinations with and without 50 µM CPZ, before the addition of PK (Fig. 6). Fragments were separated by SDS-PAGE and visualized by Western blotting using a specific antibody to the C-terminus of the α-subunit. In control samples, the C-terminal half of the α-subunit (a fragment of 55 kDa starting at an area close to the phosphorylation site in the P domain to the C-terminal end of the α-subunit), containing M5-M10, is accumulated in the presence of K+ alone, whereas in the presence of Na+ or Na+ and K+, the level of the 55 kDa fragment was largely reduced (Fig. 6, compare lanes labeled Na+, K+, and Na+ plus K+). Parallel experiments where cleavage products were stained with Coomassie blue indicated that the 55 kDa is almost completely cleaved to smaller fragment (data not shown). This indicates that the C-terminal half of enzyme incubated in the presence of Na+ alone is less stable compared to the K+-bound enzyme, and that the presence of Na+ together with K+ abrogates the protection provided by K+. In samples treated with CPZ and then subjected to PK cleavage, in contrast, the 55 kDa fragment was protected in the presence of Na+ alone or in the presence of Na+ and K+. In addition, CPZ treatment exposed a K+-specific new cleavage site that produced a 35 kDa fragment. The 35 kDa fragment is likely a result of cleavage of the 55 kDa fragment at the area connecting the N and P domains [30]. Finally, in the presence of ADP which stabilizes a compact cytoplasmic headpiece structure, the α-subunit was protected in the presence of either Na+ or K+ and CPZ still had an appreciable protecting effect.


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)

Proteinase K cleavage.Pig kidney membranes were incubated in 25/or 1 mM ADP, as indicated. The samples were treated with PK (PK/protein ratio ∼1∶50) and the mixture incubated for 40 min at 24°C, and terminated with SDS sample buffer acidified with TCA. Proteolytic products were separated using SDS-PAGE and protein fragments on the gel were transferred to PVDF membranes and visualized by Western blotting using NKA1012-1016 antibody. The labels to the left indicate the apparent molecular weights of the fragments, determined by the use of Precision plus protein standards from Bio-Rad, Cat#161-0363.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096909-g006: Proteinase K cleavage.Pig kidney membranes were incubated in 25/or 1 mM ADP, as indicated. The samples were treated with PK (PK/protein ratio ∼1∶50) and the mixture incubated for 40 min at 24°C, and terminated with SDS sample buffer acidified with TCA. Proteolytic products were separated using SDS-PAGE and protein fragments on the gel were transferred to PVDF membranes and visualized by Western blotting using NKA1012-1016 antibody. The labels to the left indicate the apparent molecular weights of the fragments, determined by the use of Precision plus protein standards from Bio-Rad, Cat#161-0363.
Mentions: Cleavage with PK of the cytoplasmic domains of the α-subunit was used as a tool to investigate the effect of CPZ on the conformation of pig kidney Na+,K+-ATPase. In the presence of occluded ions, a specific cleavage pattern is obtained as proteolytic sites at distinct locations are protected and others are exposed. In the absence of occluded ions, this protection is absent and all of the cytoplasmic domains of the α-subunit are completely cleaved. To this end, membrane-bound enzyme was incubated in the presence of different ion combinations with and without 50 µM CPZ, before the addition of PK (Fig. 6). Fragments were separated by SDS-PAGE and visualized by Western blotting using a specific antibody to the C-terminus of the α-subunit. In control samples, the C-terminal half of the α-subunit (a fragment of 55 kDa starting at an area close to the phosphorylation site in the P domain to the C-terminal end of the α-subunit), containing M5-M10, is accumulated in the presence of K+ alone, whereas in the presence of Na+ or Na+ and K+, the level of the 55 kDa fragment was largely reduced (Fig. 6, compare lanes labeled Na+, K+, and Na+ plus K+). Parallel experiments where cleavage products were stained with Coomassie blue indicated that the 55 kDa is almost completely cleaved to smaller fragment (data not shown). This indicates that the C-terminal half of enzyme incubated in the presence of Na+ alone is less stable compared to the K+-bound enzyme, and that the presence of Na+ together with K+ abrogates the protection provided by K+. In samples treated with CPZ and then subjected to PK cleavage, in contrast, the 55 kDa fragment was protected in the presence of Na+ alone or in the presence of Na+ and K+. In addition, CPZ treatment exposed a K+-specific new cleavage site that produced a 35 kDa fragment. The 35 kDa fragment is likely a result of cleavage of the 55 kDa fragment at the area connecting the N and P domains [30]. Finally, in the presence of ADP which stabilizes a compact cytoplasmic headpiece structure, the α-subunit was protected in the presence of either Na+ or K+ and CPZ still had an appreciable protecting effect.

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