Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

Background: Na+,K+-ATPase mutations extending the C terminus cause neurological disease. Results: C-terminal extension reduces Na+ affinity. Analysis of several mutants establishes a relationship between change in Na+ affinity and change of intracellular Na+ and K+ concentrations. Conclusion: The Na+ affinity of the Na+,K+-ATPase is a major in vivo determinant of the intracellular Na+ concentration. Significance: Insight in pathophysiology and regulation of the Na+,K+-ATPase is obtained. The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na+,K+-ATPase (2) and (3) isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na+,K+-ATPase mutations, extending the C terminus by either 28 residues (+28 mutation) or an extra tyrosine (+Y), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na+ and K+ concentrations ([Na+](i) and [K+](i)) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na+ affinity without disturbance of K+ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of (2) despite a high expression level. A significant rise of [Na+](i) and reduction of [K+](i) was detected in cells expressing mutants with reduced Na+ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na+ affinity were found to reduce [Na+](i). It is concluded that the Na+ affinity of the Na+,K+-ATPase is an important determinant of [Na+](i).