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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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Proteolytic cleavage site in the E1 structure of the Na+,K+-ATPase.Architecture of the cytoplasmic loop between membrane spans 6 and 7, as well as the cytoplasmic part of membrane spans 8–10 together with the C-terminal extension in the E1·AlF4−·ADP·3Na+ (PDB accession nr 3WGU). The figure was made using Pymol (www.pymol.com). D926, which coordinates Na+ in the unique site, is shown in red. Part of the intracellular loop between membrane spans 6 and 7 (L67) is shown. This part contains an asparagine residue (N831, shown in white), cleavage at which produces the C-terminal 19 kDa fragment of the α-subunit [Ref. 38]. Positive amino acids in the C-terminal part of the α-subunit are shown in blue. T4 indicates the cleavage site described in this study, occurring between R1005 and P1006, as evidenced from Edman degradation. Part of M9, shown in cyan, appears behind M8. The dashed double-headed arrow indicates hypothetical movements of M10 that would result in deflection of the C-terminal tail and consequently modification of the ion binding sites.
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pone-0096909-g012: Proteolytic cleavage site in the E1 structure of the Na+,K+-ATPase.Architecture of the cytoplasmic loop between membrane spans 6 and 7, as well as the cytoplasmic part of membrane spans 8–10 together with the C-terminal extension in the E1·AlF4−·ADP·3Na+ (PDB accession nr 3WGU). The figure was made using Pymol (www.pymol.com). D926, which coordinates Na+ in the unique site, is shown in red. Part of the intracellular loop between membrane spans 6 and 7 (L67) is shown. This part contains an asparagine residue (N831, shown in white), cleavage at which produces the C-terminal 19 kDa fragment of the α-subunit [Ref. 38]. Positive amino acids in the C-terminal part of the α-subunit are shown in blue. T4 indicates the cleavage site described in this study, occurring between R1005 and P1006, as evidenced from Edman degradation. Part of M9, shown in cyan, appears behind M8. The dashed double-headed arrow indicates hypothetical movements of M10 that would result in deflection of the C-terminal tail and consequently modification of the ion binding sites.

Mentions: We have provided direct evidence for a CPZ-mediated conformational change that exposes the C-terminal domain of the α-subunit to trypsin cleavage (as shown in Fig. 8) in the presence of Gua+, an ion that was previously shown to permeate the Na+ specific site [36], [37]. Fig. 12 shows the location of the T4 site in the crystal structure of the pig kidney enzyme in the E1·AlF4-·ADP·3Na+ form (PDB accession nr 3WGU, Ref. [9]). The three C-terminal arginine residues, R1003-R1005, build the last C-terminal turn in M10. A similar architecture is also present in the E2·MgF42-·2K+ structure [7]. Proteolytic data (Figs. 7 and 8) suggest that the distal part of M10 is buried in the membrane but is exposed to trypsin attack by a conformational change. The major factor that produces exposure of the C-terminal tail was found to be Gua+. The effect of Gua+ is enhanced by either high pH (Fig. 7) or CPZ (Fig. 8). Surprisingly, the effects seen in the presence of Gua+ were not reproduced by Na+. A similar paradox was indeed reported in the study by Ratheal et al. [37]; H+ and Gua+ were found to leak through site III in the pump whereas Na+ did not. This is likely related to the fact that Na+ can bind both the shared and the specific site whereas Gua+ cannot. If binding to the shared sites controls access to the Na+ specific site, the unique effect of Gua+ may be reconciled.


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)

Proteolytic cleavage site in the E1 structure of the Na+,K+-ATPase.Architecture of the cytoplasmic loop between membrane spans 6 and 7, as well as the cytoplasmic part of membrane spans 8–10 together with the C-terminal extension in the E1·AlF4−·ADP·3Na+ (PDB accession nr 3WGU). The figure was made using Pymol (www.pymol.com). D926, which coordinates Na+ in the unique site, is shown in red. Part of the intracellular loop between membrane spans 6 and 7 (L67) is shown. This part contains an asparagine residue (N831, shown in white), cleavage at which produces the C-terminal 19 kDa fragment of the α-subunit [Ref. 38]. Positive amino acids in the C-terminal part of the α-subunit are shown in blue. T4 indicates the cleavage site described in this study, occurring between R1005 and P1006, as evidenced from Edman degradation. Part of M9, shown in cyan, appears behind M8. The dashed double-headed arrow indicates hypothetical movements of M10 that would result in deflection of the C-terminal tail and consequently modification of the ion binding sites.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096909-g012: Proteolytic cleavage site in the E1 structure of the Na+,K+-ATPase.Architecture of the cytoplasmic loop between membrane spans 6 and 7, as well as the cytoplasmic part of membrane spans 8–10 together with the C-terminal extension in the E1·AlF4−·ADP·3Na+ (PDB accession nr 3WGU). The figure was made using Pymol (www.pymol.com). D926, which coordinates Na+ in the unique site, is shown in red. Part of the intracellular loop between membrane spans 6 and 7 (L67) is shown. This part contains an asparagine residue (N831, shown in white), cleavage at which produces the C-terminal 19 kDa fragment of the α-subunit [Ref. 38]. Positive amino acids in the C-terminal part of the α-subunit are shown in blue. T4 indicates the cleavage site described in this study, occurring between R1005 and P1006, as evidenced from Edman degradation. Part of M9, shown in cyan, appears behind M8. The dashed double-headed arrow indicates hypothetical movements of M10 that would result in deflection of the C-terminal tail and consequently modification of the ion binding sites.
Mentions: We have provided direct evidence for a CPZ-mediated conformational change that exposes the C-terminal domain of the α-subunit to trypsin cleavage (as shown in Fig. 8) in the presence of Gua+, an ion that was previously shown to permeate the Na+ specific site [36], [37]. Fig. 12 shows the location of the T4 site in the crystal structure of the pig kidney enzyme in the E1·AlF4-·ADP·3Na+ form (PDB accession nr 3WGU, Ref. [9]). The three C-terminal arginine residues, R1003-R1005, build the last C-terminal turn in M10. A similar architecture is also present in the E2·MgF42-·2K+ structure [7]. Proteolytic data (Figs. 7 and 8) suggest that the distal part of M10 is buried in the membrane but is exposed to trypsin attack by a conformational change. The major factor that produces exposure of the C-terminal tail was found to be Gua+. The effect of Gua+ is enhanced by either high pH (Fig. 7) or CPZ (Fig. 8). Surprisingly, the effects seen in the presence of Gua+ were not reproduced by Na+. A similar paradox was indeed reported in the study by Ratheal et al. [37]; H+ and Gua+ were found to leak through site III in the pump whereas Na+ did not. This is likely related to the fact that Na+ can bind both the shared and the specific site whereas Gua+ cannot. If binding to the shared sites controls access to the Na+ specific site, the unique effect of Gua+ may be reconciled.

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