The Na,K-ATPase provides the driving force for many ion transport processes through control of Na ؉ and K ؉ concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, ␣ and ␤. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the ␥-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the ␥-subunit and to phospholemman (PLM) was identified in purified Na,KATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na,K-ATPase ␣-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the ␣-subunit of pig kidney Na,K-ATPase, indicating specific association with different ␣-isoforms. Finally, PLMS and the ␣-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with nonsolubilizing concentrations of C 12 E 8 resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.

Proteins of the FXYD family act as tissue-specific regulators of the Na-K-ATPase. They are small hydrophobic type I proteins with a single-transmembrane span containing an extracellular invariant FXYD sequence. FXYD proteins are not an integral part of the Na-K-ATPase but function to modulate its catalytic properties by molecular interactions with specific Na-K-ATPase domains.

Background:The ␣2 isoform of Na,K-ATPase is unstable compared with ␣1 and ␣3. Results: Mutations in TM8 -10 strongly stabilize ␣2. A novel phospholipid antagonist selectively inactivates ␣2, and mutations in TM8 -10 protect against inactivation. Conclusion: A phosphatidylserine binding pocket within TM8 -10 has been identified. Significance: Mechanistic insights into ␣2 instability and a possible physiological role have been obtained.

Glutathionylation of cysteine 46 of the β1 subunit of the Na+-K+ pump causes pump inhibition. However, the crystal structure, known in a state analogous to an E2·2K+·Pi configuration, indicates that the side chain of cysteine 46 is exposed to the lipid bulk phase of the membrane and not expected to be accessible to the cytosolic glutathione. We have examined whether glutathionylation depends on the conformational changes in the Na+-K+ pump cycle as described by the Albers-Post scheme. We measured β1 subunit glutathionylation and function of Na+-K+-ATPase in membrane fragments and in ventricular myocytes. Signals for glutathionylation in Na+-K+-ATPase-enriched membrane fragments suspended in solutions that preferentially induce E1ATP and E1Na3 conformations were much larger than signals in solutions that induce the E2 conformation. Ouabain further reduced glutathionylation in E2 and eliminated an increase seen with exposure to the oxidant peroxynitrite (ONOO−). Inhibition of Na+-K+-ATPase activity after exposure to ONOO− was greater when the enzyme had been in the E1Na3 than the E2 conformation. We exposed myocytes to different extracellular K+ concentrations to vary the membrane potential and hence voltage-dependent conformational poise. K+ concentrations expected to shift the poise toward E2 species reduced glutathionylation, and ouabain eliminated a ONOO−-induced increase. Angiotensin II-induced NADPH oxidase-dependent Na+-K+ pump inhibition was eliminated by conditions expected to shift the poise toward the E2 species. We conclude that susceptibility of the β1 subunit to glutathionylation depends on the conformational poise of the Na+-K+ pump.

Background and purpose: Previous studies have identified the natural polyphenol curcumin as a protein kinase C (PKC) inhibitor. In contrast, we found significant stimulation of PKC activity following curcumin treatment. Thus, the mechanism of curcumin interaction with PKC was investigated. Experimental approach: We employed phosphorylation assays in the presence of soluble or membrane-bound PKC substrates, followed by SDS-PAGE, autoradiography and phosphorylation intensity measurements. Key results: Curcumin inhibited PKC in the absence of membranes whereas stimulation was observed in the presence of membranes. Further analysis indicated that curcumin decreased PKC activity by competition with Ca 2 þ stimulation of the kinase, resulting in inhibition of activity at lower Ca 2 þ concentrations and stimulation at higher Ca 2 þ concentrations. The role of the membrane is likely to be facilitation of Ca 2 þ -binding to the kinase, thus relieving the curcumin inhibition observed at limited Ca 2 þ concentrations. Curcumin was found to mildly stimulate the catalytic subunit of PKC, which does not require Ca 2 þ for activation. In addition, studies on Ca 2 þ -independent PKC isoforms as well as another curcumin target (the sarcoplasmic reticulum Ca 2 þ -ATPase) confirmed a correlation between Ca 2 þ concentration and the curcumin effects. Conclusions and Implications: Curcumin competes with Ca 2 þ for the regulatory domain of PKC, resulting in a Ca 2 þ -dependent dual effect on the kinase. We propose that curcumin interacts with the Ca 2 þ -binding domains in target proteins. To our knowledge, this is the first study that defines an interaction domain for curcumin, and provides a rationale for the broad specificity of this polyphenol as a chemopreventive drug.