CCCP activation of the reconstituted NaK-pump

Abstract

In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na+ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15 degrees C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (Vm), we postulated that a Vm build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K+ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331-355). Although cytoplasmic K+ accelerated the change of ADP- and K(+)-sensitive EP (E*P) to K(+)-sensitive ADP-insensitive EP (E2P), CCCP did not complete with cytoplasmic K+ when cytoplasmic Na+ was saturated. When the PLs were phosphorylated with 20 microM ATP and 20 microM palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E*P content and decreased the ADP-sensitive K(+)-insensitive EP (E1P). The results described above suggest that CCCP affects the E1P to E*P change in the E1P----E*P----E2P conversion and that this reaction step is inhibited by Vm.

The Rapid-onset Dystonia Parkinsonism Mutation D923N of the Na+,K+-ATPase α3 Isoform Disrupts Na+ Interaction at the Third Na+ Site

“…Hence, the results generated by the computation can be considered rather accurate. Na ϩ is released at the extracellular side of the membrane in connection with the E 1 P 3 E 2 P conformational transition (Scheme 1), and the binding of extracellular Na ϩ reverses this transition in the wild type (32,33), thus promoting accumulation of E 1 P. Hence, the dephosphorylation data are consistent with the hypothesis that one or more external Na ϩ site(s) is affected. Because the Na ϩ activation of E 2 P dephosphorylation was not disturbed by the mutation, the affected external site is probably not one of those binding K ϩ during the Na ϩ ,K ϩ -ATPase cycle, which is in good accordance with the conclusion drawn above that K ϩ interaction is normal in the mutant.…”

“…The slightly higher rate of the mutant may be explained by a higher amount of E 2 P accumulated during the phosphorylation (see below and Table 1). In the absence of K ϩ , Na ϩ at a high concentration has dual effects on dephosphorylation in the wild type; (i) Na ϩ , bound with low affinity at the external sites of E 2 P, is able to mimic K ϩ , thus activating dephosphorylation to some extent and being transported toward the cytoplasmic side at a stoichiometry of two Na ϩ per ATP hydrolyzed (30,31), and (ii) Na ϩ bound at an external site(s) reverses the E 1 P 3 E 2 P conformational transition, promoting accumulation of E 1 P (32, 33). In the wild type the net result of these two opposite effects is that the rate of dephosphorylation is higher at 600 mM Na ϩ compared with 20 mM Na ϩ .…”

The C Terminus of Na+,K+-ATPase Controls Na+ Affinity on Both Sides of the Membrane through Arg935

“…Na ϩ is released at the extracellular side of the membrane in association with the conformational transition from E 1 P to E 2 P, and a high extracellular Na ϩ concentration drives the E 1 P 3 E 2 P transition backwards, resulting in accumulation of E 1 P that can be dephosphorylated by ADP (21,22). To examine whether the increased E 2 P fraction of the phosphoenzyme seen for some of the mutants at 150 mM Na ϩ is caused by a reduced ability of the phosphoenzyme to bind extracellular Na ϩ , we studied the interaction of Na ϩ with the extracellularly facing low affinity sites further in selected mutants, ⌬WVEKETYY, YY-AA, and K768A.…”

“…Under these conditions, the maximal catalytic turnover rate is only a small percentage of that obtained in the presence of K ϩ because Na ϩ is much less efficient as an activator of E 2 P dephosphorylation as compared with K ϩ (3). Extracellular Na ϩ at high concentrations (K 0.5 (ligand concentration giving half-maximum effect) 600 -700 mM) inhibits the Na ϩ -ATPase activity of the wild type by displacing the E 1 P-E 2 P conformational equilibrium in favor of E 1 P (21,22). Again, this is a rather specific effect of Na ϩ because choline chloride in the same concentration range exerted much less inhibition (see supplemental Fig.…”

Molecular imaging of Na+,K+‐ATPase in purified kidney membranes

“…Recent studies employing recombinant DNA techniques have identified distinct ion-sensitive domains, but the mechanism of transport of these ions across the membrane remains to be solved [16,[21][22][23][24]. Since a marine toxin, palytoxin, is known to increase ouabain-sensitive Na ÷ conductance to 9.6 pS which is comparable to conductance values obtained for known ion channels [25,26], it might be that the Na÷,K÷-ATPase takes a specific channel-like conformation under a certain condition. Our present data on the molecular structure of the Na÷,K÷-ATPase (Fig.…”