Na,K-ATPase Isozymes in Colorectal Cancer and Liver Metastases

The cell membrane Na,K-ATPase is a member of the P-type family of active cation transport proteins. Recently the molecular structure of the related sarcoplasmic reticulum Ca-ATPase in an E1 conformation has been determined at 2.6 A resolution. Furthermore, theoretical models of the Ca-ATPase in E2 conformations are available. As a result of these developments, these structural data have allowed construction of homology models that address the central questions of mechanism of active cation transport by all P-type cation pumps. This review relates recent evidence on functional sites of Na,K-ATPase for the substrate (ATP), the essential cofactor (Mg(2+) ions), and the transported cations (Na(+) and K(+)) to the molecular structure. The essential elements of the Ca-ATPase structure, including 10 transmembrane helices and well-defined N, P, and A cytoplasmic domains, are common to all PII-type pumps such as Na,K-ATPase and H,K-ATPases. However, for Na,K-ATPase and H,K-ATPase, which consist of both alpha- and beta-subunits, there may be some detailed differences in regions of subunit interactions. Mutagenesis, proteolytic cleavage, and transition metal-catalyzed oxidative cleavages are providing much evidence about residues involved in binding of Na(+), K(+), ATP, and Mg(2+) ions and changes accompanying E1-E2 or E1-P-E2-P conformational transitions. We discuss this evidence in relation to N, P, and A cytoplasmic domain interactions, and long-range interactions between the active site and the Na(+) and K(+) sites in the transmembrane segments, for the different steps of the catalytic cycle.

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 alpha and beta isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K(+)-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the alpha isoform. On the other hand, variations in external K(+) activation are determined by a cooperative interaction mechanism between alpha and beta isoforms with alpha2-beta2 complexes having the lowest apparent K(+) affinity. alpha Isoforms influence the apparent internal Na(+) affinity in the order alpha1 > alpha2 > alpha3 and the voltage dependence in the order alpha2 > alpha1 > alpha3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, alpha2-beta isozymes exhibit more rapid ouabain association as well as dissociation rate constants than alpha1-beta and alpha3-beta isozymes. Finally, isoform-specific differences exist in the K(+)/ouabain antagonism which may protect alpha1 but not alpha2 or alpha3 from digitalis inhibition at physiological K(+) levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.