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

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 (?(m)=0.2-0.5?ms) and slow (?(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.

SUBMITTER: Gadsby DC

PROVIDER: S-EPMC3293407 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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

Gadsby David C DC Bezanilla Francisco F Rakowski Robert F RF De Weer Paul P Holmgren Miguel M

Nature communications 20120214

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 th ...[more]

PMID: 22334072

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Project description:The whole-cell voltage-clamp technique was used in rat cardiac myocytes to investigate the kinetics of ADP binding to phosphorylated states of Na,K-ATPase and its effects on presteady-state Na(+)-dependent charge movements by this enzyme. Ouabain-sensitive transient currents generated by Na,K-ATPase functioning in electroneutral Na(+)-Na(+) exchange mode were measured at 23 degrees C with pipette ADP concentrations ([ADP]) of up to 4.3 mM and extracellular Na(+) concentrations ([Na](o)) between 36 and 145 mM at membrane potentials (V(M)) from -160 to +80 mV. Analysis of charge-V(M) curves showed that the midpoint potential of charge distribution was shifted toward more positive V(M) both by increasing [ADP] at constant Na(+)(o) and by increasing [Na](o) at constant ADP. The total quantity of mobile charge, on the other hand, was found to be independent of changes in [ADP] or [Na](o). The presence of ADP increased the apparent rate constant for current relaxation at hyperpolarizing V(M) but decreased it at depolarizing V(M) as compared to control (no added ADP), an indication that ADP binding facilitates backward reaction steps during Na(+)-Na(+) exchange while slowing forward reactions. Data analysis using a pseudo three-state model yielded an apparent K(d) of approximately 6 mM for ADP binding to and release from the Na,K-ATPase phosphoenzyme; a value of 130 s(-1) for k(2), a rate constant that groups Na(+) deocclusion/release and the enzyme conformational transition E(1) approximately P --> E(2)-P; a value of 162 s(-1)M(-1) for k(-2), a lumped second-order V(M)-independent rate constant describing the reverse reactions; and a Hill coefficient of approximately 1 for Na(+)(o) binding to E(2)-P. The results are consistent with electroneutral release of ADP before Na(+) is deoccluded and released through an ion well. The same approach can be used to study additional charge-moving reactions and associated electrically silent steps of the Na,K-pump and other transporters.

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

Publications

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

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