Inhibition of phosphorylation of na+,k+-ATPase by mutations causing familial hemiplegic migraine.

The neurological disorder familial hemiplegic migraine type II (FHM2) is caused by mutations in the α2-isoform of the Na(+),K(+)-ATPase. We have studied the partial reaction steps of the Na(+),K(+)-pump cycle in nine FHM2 mutants retaining overall activity at a level still compatible with cell growth. Although it is believed that the pathophysiology of FHM2 results from reduced extracellular K(+) clearance and/or changes in Na(+) gradient-dependent transport processes in neuroglia, a reduced affinity for K(+) or Na(+) is not a general finding with the FHM2 mutants. Six of the FHM2 mutations markedly affect the maximal rate of phosphorylation from ATP leading to inhibition by intracellular K(+), thereby likely compromising pump function under physiological conditions. In mutants R593W, V628M, and M731T, the defective phosphorylation is caused by local perturbations within the Rossmann fold, possibly interfering with the bending of the P-domain during phosphoryl transfer. In mutants V138A, T345A, and R834Q, long range effects reaching from as far away as the M2 transmembrane helix perturb the function of the catalytic site. Mutant E700K exhibits a reduced rate of E(2)P dephosphorylation without effect on phosphorylation from ATP. An extremely reduced vanadate affinity of this mutant indicates that the slow dephosphorylation reflects a destabilization of the phosphoryl transition state. This seems to be caused by insertion of the lysine between two other positively charged residues of the Rossmann fold. In mutants R202Q and T263M, effects on the A-domain structure are responsible for a reduced rate of the E(1)P to E(2)P transition.

The C terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935.

The Na(+),K(+)-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na(+) sites, as well as E(1)-E(2) conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr(1017) and Tyr(1018), as well as putative interaction partners, Arg(935) in the loop between transmembrane segments M8 and M9 and Lys(768) in transmembrane segment M5, as crucial to Na(+) activation of phosphorylation of E(1), a partial reaction reflecting Na(+) interaction on the cytoplasmic side of the membrane. Tyr(1017), Tyr(1018), and Arg(935) are furthermore indispensable to Na(+) interaction on the extracellular side of the membrane, as revealed by inability of high Na(+) concentrations to drive the transition from E(1)P to E(2)P backwards toward E(1)P and inhibit Na(+)-ATPase activity in mutants. Lys(768) is not important for Na(+) binding from the external side of the membrane but is involved in stabilization of the E(2) form. These data demonstrate that the C terminus controls Na(+) affinity on both sides of the membrane and suggest that Arg(935) constitutes an important link between the C terminus and the third Na(+) site, involving an arginine-pi stacking interaction between Arg(935) and the C-terminal tyrosines. Lys(768) may interact preferentially with the C terminus in E(1) and E(1)P forms and with the loop between transmembrane segments M6 and M7 in E(2) and E(2)P forms.

The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations.

The Na+,K+-ATPase transforms the energy of ATP to the maintenance of steep electrochemical gradients for sodium and potassium across the plasma membrane. This activity is tissue specific, in particular due to variations in the expressions of the alpha subunit isoforms one through four. Several mutations in alpha2 and 3 have been identified that link the specific function of the Na+,K+-ATPase to the pathophysiology of neurological diseases such as rapid-onset dystonia parkinsonism and familial hemiplegic migraine type 2. We show a mapping of the isoform differences and the disease-related mutations on the recently determined crystal structure of the pig renal Na+,K+-ATPase and a structural comparison to Ca2+-ATPase. Furthermore, we present new experimental data that address the role of a stretch of three conserved arginines near the C-terminus of the alpha subunit (Arg1003-Arg1005).

Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase.

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.