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Interaction of the alpha subunit of Na,K-ATPase with cofilin.

ABSTRACT: The alpha1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the alpha subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive (86)Rb(+) uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.

SUBMITTER: Lee K

PROVIDER: S-EPMC1221581 | biostudies-other | 2001 Jan

REPOSITORIES: biostudies-other

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Project description:The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.

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Interaction of the alpha subunit of Na,K-ATPase with cofilin.

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