Opposing Effects of Na⁺ and K⁺ on the Thermal Stability of Na⁺,K⁺-ATPase

Abstract

Folding and structural stability are key factors for the proper biological function of proteins. Na(+),K(+)-ATPase is an integral membrane protein involved in the active transport of Na(+) and K(+) across the plasma membrane. In this work we characterized the effects of K(+) and Na(+) on the thermal inactivation of Na(+),K(+)-ATPase, evaluating both catalytic and transport capacities of the pump. Both activities of the enzyme decrease with the preincubation time as first-order kinetics. The thermal inactivation of Na(+),K(+)-ATPase is simultaneous with a conformational change detected by tryptophan and 1-aniline-8-naphtalenesulfonate (ANS) fluorescence. The kinetic coefficient of thermal inactivation was affected by the presence of Na(+) and K(+) (or Rb(+)) and the temperature of the preincuabtion media. Our results show that K(+) or Rb(+) stabilize the enzyme, while Na(+) decreases the stability of Na(+),K(+)-ATPase. Both effects are exerted by the specific binding of these cations to the pump. Also, we provided strong evidence that the Rb(+) (or K(+)) stabilization effect is due to the occlusion of these cations into the enzyme. Here, we proposed a minimal kinetic model that explains the behavior observed in the experimental results and allows a better understanding of the results presented by other researchers. The thermal inactivation process was also analyzed according to Kramer's theory.

Kinetic stability of membrane proteins

“…This was the case of several P-ATPases for which the kinetic coefficients describing the time course of the structural changes detected by Trp, ANS and eosin fluorescence, circular dichroism, and ion occlusion capacity are not statistically different from that corresponding to the inactivation process (Cattoni et al 2008;Kaufman et al 2012;Levi et al 2000;Placenti et al 2017), indicating that changes in the enzyme activity and secondary and tertiary structure are all part of the same global change produced by incubation at high temperatures.…”

“…In P-type ATPases, it was found that the decrease in enzyme activity is paralleled by large changes in secondary and tertiary structure (Cattoni et al 2008;Kaufman et al 2012;Levi et al 2000;Placenti et al 2017). The irreversible loss of ATPase activity was not due to either degradation or formation of SDS-stable aggregates of the protein.…”

“…This model was successfully used to explain the effects of several ligands on the Na + ,K + -ATPase thermal stability (Kaufman et al 2012;Placenti et al 2017).…”

“…In the Na + ,K + -ATPase, the ΔG ‡ value measured in the absence of ligands (56 kJ/mol) arises from large positive differences in enthalpic (476 kJ/mol) and entropic (420 kJ/mol) contributions (Kaufman et al 2012). Different enzyme ligands (Na + , K + , Mg 2+ , ATP) produced a slight change in the activation free energy, which is positive for K + , Mg 2+ , and ATP (Placenti et al 2017) and negative for Na + (Kaufman et al 2012).…”

“…In the Na + ,K + -ATPase, the ΔG ‡ value measured in the absence of ligands (56 kJ/mol) arises from large positive differences in enthalpic (476 kJ/mol) and entropic (420 kJ/mol) contributions (Kaufman et al 2012). Different enzyme ligands (Na + , K + , Mg 2+ , ATP) produced a slight change in the activation free energy, which is positive for K + , Mg 2+ , and ATP (Placenti et al 2017) and negative for Na + (Kaufman et al 2012). The destabilizing effect of Na + was explained because this cation shifted the conformational equilibrium of the enzyme toward a less stable conformation, denoted as E 1 (Kaufman et al 2012;Yoneda et al 2013).…”