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Effect of ADP on Na⁺-Na⁺ Exchange Reaction Kinetics of Na,K-ATPase

Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07101

Correspondence: Address reprint requests to Dr. R. Daniel Peluffo, Dept. of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, 185 S. Orange Ave., PO Box 1709, Newark, NJ 07101-1709. Tel.: 973-972-1490; Fax: 973-972-7950; E-mail: peluffrd{at}umdnj.edu.

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°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 6 mM for ADP binding to and release from the Na,K-ATPase phosphoenzyme; a value of 130 s^–1 for k₂, a rate constant that groups Na⁺ deocclusion/release and the enzyme conformational transition E₁P E₂-P; a value of 162 s^–1M^–1 for k_–2, a lumped second-order V_M-independent rate constant describing the reverse reactions; and a Hill coefficient of 1 for Na⁺_o binding to E₂-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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