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The dynamic relationships between the three events that release individual Na⁺ ions from the Na⁺/K⁺-ATPase.

Gadsby DC, Bezanilla F, Rakowski RF, De Weer P, Holmgren M - Nat Commun (2012)

Bottom Line: By applying rapid voltage steps to squid giant axons, we previously identified three components in such transient currents, with distinct relaxation speeds: fast (which nearly parallels the voltage-jump time course), medium speed (τ(m)=0.2-0.5 ms) and slow (τ(s)=1-10 ms).Here we show that these three components are tightly correlated, both in their magnitudes and in the time courses of their changes.The correlations reveal the dynamics of the conformational rearrangements that release three Na(+) to the exterior (or sequester them into their binding sites) one at a time, in an obligatorily sequential manner.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA. gadsby@rockefeller.edu

ABSTRACT
Na(+)/K(+) pumps move net charge through the cell membrane by mediating unequal exchange of intracellular Na(+) and extracellular K(+). Most charge moves during transitions that release Na(+) to the cell exterior. When pumps are constrained to bind and release only Na(+), a membrane voltage-step redistributes pumps among conformations with zero, one, two or three bound Na(+), thereby transiently generating current. By applying rapid voltage steps to squid giant axons, we previously identified three components in such transient currents, with distinct relaxation speeds: fast (which nearly parallels the voltage-jump time course), medium speed (τ(m)=0.2-0.5 ms) and slow (τ(s)=1-10 ms). Here we show that these three components are tightly correlated, both in their magnitudes and in the time courses of their changes. The correlations reveal the dynamics of the conformational rearrangements that release three Na(+) to the exterior (or sequester them into their binding sites) one at a time, in an obligatorily sequential manner.

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Post-Albers diagram of the Na+/K+ transport cycle.In this simplified scheme, E1 conformations represent those in which binding sites may be accessed from the intracellular side. In these states, two K+ (orange balls) are released, three Na+ (green balls) are bound and occluded, and the protein is phosphorylated. In P-E2 conformations, the binding sites become accessible from the external environment, and the three Na+ are released, two K+ are bound and occluded, and the protein autodephosphorylates. The states enclosed by the dotted box were isolated by removal of K+ from all solutions and by the presence of 5 mM MgATP (and of ADP scavengers)814 in the internal solution.
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f1: Post-Albers diagram of the Na+/K+ transport cycle.In this simplified scheme, E1 conformations represent those in which binding sites may be accessed from the intracellular side. In these states, two K+ (orange balls) are released, three Na+ (green balls) are bound and occluded, and the protein is phosphorylated. In P-E2 conformations, the binding sites become accessible from the external environment, and the three Na+ are released, two K+ are bound and occluded, and the protein autodephosphorylates. The states enclosed by the dotted box were isolated by removal of K+ from all solutions and by the presence of 5 mM MgATP (and of ADP scavengers)814 in the internal solution.

Mentions: On the basis of biochemical data accumulated during the decade following its discovery1, the Na+/K+-ATPase was proposed to transport Na+ and K+ ions according to a model known as the Post-Albers scheme23 (Fig. 1). As ions are transported through the Na+/K+ pump, they become temporarily occluded within the protein, inaccessible (symbolized by parentheses) from either cytoplasmic or extracellular side of the membrane, before being released4567. By working in the absence of intracellular and extracellular K+, but presence of intracellular ATP, thereby effectively restricting Na+/K+ pumps to the reversible transitions associated with deocclusion/occlusion and extracellular release/binding of Na+ (conformations encompassed by the dotted box in Fig. 1), Nakao and Gadsby8 were able to detect presteady-state electrical signals accompanying those transitions in response to steps of membrane potential. The signals arise because Na+ ions traverse a fraction of the transmembrane electric field as they enter or leave their binding sites deep within the pump910111213. At a given membrane potential and external sodium concentration ([Na+]o), the populations of pumps with empty binding sites, and those with bound or occluded Na+ (that is, dotted box conformations, Fig. 1), reach a steady-state distribution. A sudden change of membrane voltage then shifts the Na+-binding equilibrium, and initiates a redistribution of the pump populations towards a new steady-state arrangement. The consequent change in Na+-binding-site occupancy causes Na+ ions to travel between the extracellular environment and the pump interior, through the electric field, so generating current. As the new steady distribution is approached, fewer Na+ ions move, and the current declines. The electrical signals therefore appear as transient currents.


The dynamic relationships between the three events that release individual Na⁺ ions from the Na⁺/K⁺-ATPase.

Gadsby DC, Bezanilla F, Rakowski RF, De Weer P, Holmgren M - Nat Commun (2012)

Post-Albers diagram of the Na+/K+ transport cycle.In this simplified scheme, E1 conformations represent those in which binding sites may be accessed from the intracellular side. In these states, two K+ (orange balls) are released, three Na+ (green balls) are bound and occluded, and the protein is phosphorylated. In P-E2 conformations, the binding sites become accessible from the external environment, and the three Na+ are released, two K+ are bound and occluded, and the protein autodephosphorylates. The states enclosed by the dotted box were isolated by removal of K+ from all solutions and by the presence of 5 mM MgATP (and of ADP scavengers)814 in the internal solution.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293407&req=5

f1: Post-Albers diagram of the Na+/K+ transport cycle.In this simplified scheme, E1 conformations represent those in which binding sites may be accessed from the intracellular side. In these states, two K+ (orange balls) are released, three Na+ (green balls) are bound and occluded, and the protein is phosphorylated. In P-E2 conformations, the binding sites become accessible from the external environment, and the three Na+ are released, two K+ are bound and occluded, and the protein autodephosphorylates. The states enclosed by the dotted box were isolated by removal of K+ from all solutions and by the presence of 5 mM MgATP (and of ADP scavengers)814 in the internal solution.
Mentions: On the basis of biochemical data accumulated during the decade following its discovery1, the Na+/K+-ATPase was proposed to transport Na+ and K+ ions according to a model known as the Post-Albers scheme23 (Fig. 1). As ions are transported through the Na+/K+ pump, they become temporarily occluded within the protein, inaccessible (symbolized by parentheses) from either cytoplasmic or extracellular side of the membrane, before being released4567. By working in the absence of intracellular and extracellular K+, but presence of intracellular ATP, thereby effectively restricting Na+/K+ pumps to the reversible transitions associated with deocclusion/occlusion and extracellular release/binding of Na+ (conformations encompassed by the dotted box in Fig. 1), Nakao and Gadsby8 were able to detect presteady-state electrical signals accompanying those transitions in response to steps of membrane potential. The signals arise because Na+ ions traverse a fraction of the transmembrane electric field as they enter or leave their binding sites deep within the pump910111213. At a given membrane potential and external sodium concentration ([Na+]o), the populations of pumps with empty binding sites, and those with bound or occluded Na+ (that is, dotted box conformations, Fig. 1), reach a steady-state distribution. A sudden change of membrane voltage then shifts the Na+-binding equilibrium, and initiates a redistribution of the pump populations towards a new steady-state arrangement. The consequent change in Na+-binding-site occupancy causes Na+ ions to travel between the extracellular environment and the pump interior, through the electric field, so generating current. As the new steady distribution is approached, fewer Na+ ions move, and the current declines. The electrical signals therefore appear as transient currents.

Bottom Line: By applying rapid voltage steps to squid giant axons, we previously identified three components in such transient currents, with distinct relaxation speeds: fast (which nearly parallels the voltage-jump time course), medium speed (τ(m)=0.2-0.5 ms) and slow (τ(s)=1-10 ms).Here we show that these three components are tightly correlated, both in their magnitudes and in the time courses of their changes.The correlations reveal the dynamics of the conformational rearrangements that release three Na(+) to the exterior (or sequester them into their binding sites) one at a time, in an obligatorily sequential manner.

View Article: PubMed Central - PubMed

Affiliation: Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA. gadsby@rockefeller.edu

ABSTRACT
Na(+)/K(+) pumps move net charge through the cell membrane by mediating unequal exchange of intracellular Na(+) and extracellular K(+). Most charge moves during transitions that release Na(+) to the cell exterior. When pumps are constrained to bind and release only Na(+), a membrane voltage-step redistributes pumps among conformations with zero, one, two or three bound Na(+), thereby transiently generating current. By applying rapid voltage steps to squid giant axons, we previously identified three components in such transient currents, with distinct relaxation speeds: fast (which nearly parallels the voltage-jump time course), medium speed (τ(m)=0.2-0.5 ms) and slow (τ(s)=1-10 ms). Here we show that these three components are tightly correlated, both in their magnitudes and in the time courses of their changes. The correlations reveal the dynamics of the conformational rearrangements that release three Na(+) to the exterior (or sequester them into their binding sites) one at a time, in an obligatorily sequential manner.

Show MeSH