Mads S. Toustrup-Jensen

Crystal structure of the sodium-potassium pump.

The Na+,K+-ATPase generates electrochemical gradients for sodium and potassium that are vital to animal cells, exchanging three sodium ions for two potassium ions across the plasma membrane during each cycle of ATP hydrolysis. Here we present the X-ray crystal structure at 3.5 Å resolution of the pig renal Na+,K+-ATPase with two rubidium ions bound (as potassium congeners) in an occluded state in the transmembrane part of the α-subunit. Several of the residues forming the cavity for rubidium/potassium occlusion in the Na+,K+-ATPase are homologous to those binding calcium in the Ca2+-ATPase of sarco(endo)plasmic reticulum. The β- and γ-subunits specific to the Na+,K+-ATPase are associated with transmembrane helices αM7/αM10 and αM9, respectively. The γ-subunit corresponds to a fragment of the V-type ATPase c subunit. The carboxy terminus of the α-subunit is contained within a pocket between transmembrane helices and seems to be a novel regulatory element controlling sodium affinity, possibly influenced by the membrane potential.

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).

Mutations Phe785Leu and Thr618Met in Na+,K+-ATPase, Associated with Familial Rapid-onset Dystonia Parkinsonism, Interfere with Na+ Interaction by Distinct Mechanisms

The Na+,K+-ATPase plays key roles in brain function. Recently, missense mutations in the Na+,K+-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). Here, we have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met. Both mutations lead to functionally altered, but active, Na+,K+-pumps, that display reduced apparent affinity for cytoplasmic Na+, but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E1 form with Na+ is defective, and the E1-E2 equilibrium is not displaced. In Thr618Met, the Na+ affinity is reduced because of displacement of the conformational equilibrium in favor of the K+-occluded E2(K2) form. In both mutants, K+ interaction at the external activating sites of the E2P phosphoenzyme is normal. The change of cellular Na+ homeostasis is likely a major factor contributing to the development of FRDP in patients carrying the Phe785Leu or Thr618Met mutation. Phe785Leu moreover interferes with Na+ interaction on the extracellular side and reduces the affinity for ouabain significantly. Analysis of two additional Phe785 mutants, Phe785Leu/Leu786Phe and Phe785Tyr, demonstrated that the aromatic function of the side chain, as well as its exact position, is critical for Na+ and ouabain binding. The effects of substituting Phe785 could be explained by structural modeling, demonstrating that Phe785 participates in a hydrophobic network between three transmembrane segments. Thr618 is located in the cytoplasmic part of the molecule near the catalytic site, and the structural modeling indicates that the Thr618Met mutation interferes with the bonding pattern in the catalytic site in the E1 form, thereby destabilizing E1 relative to E2(K2).

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 E1-E2 conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr1017 and Tyr1018, as well as putative interaction partners, Arg935 in the loop between transmembrane segments M8 and M9 and Lys768 in transmembrane segment M5, as crucial to Na+ activation of phosphorylation of E1, a partial reaction reflecting Na+ interaction on the cytoplasmic side of the membrane. Tyr1017, Tyr1018, and Arg935 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 E1P to E2P backwards toward E1P and inhibit Na+-ATPase activity in mutants. Lys768 is not important for Na+ binding from the external side of the membrane but is involved in stabilization of the E2 form. These data demonstrate that the C terminus controls Na+ affinity on both sides of the membrane and suggest that Arg935 constitutes an important link between the C terminus and the third Na+ site, involving an arginine-π stacking interaction between Arg935 and the C-terminal tyrosines. Lys768 may interact preferentially with the C terminus in E1 and E1P forms and with the loop between transmembrane segments M6 and M7 in E2 and E2P forms.

A C-terminal mutation of ATP1A3 underscores the crucial role of sodium affinity in the pathophysiology of rapid-onset dystonia-parkinsonism

The Na(+)/K(+)-ATPases are ion pumps of fundamental importance in maintaining the electrochemical gradient essential for neuronal survival and function. Mutations in ATP1A3 encoding the alpha3 isoform cause rapid-onset dystonia-parkinsonism (RDP). We report a de novo ATP1A3 mutation in a patient with typical RDP, consisting of an in-frame insertion of a tyrosine residue at the very C terminus of the Na(+)/K(+)-ATPase alpha3-subunit-the first reported RDP mutation in the C terminus of the protein. Expression studies revealed that there is no defect in the biogenesis or plasma membrane targeting, although cells expressing the mutant protein showed decreased survival in response to ouabain challenge. Functional analysis demonstrated a drastic reduction in Na(+) affinity in the mutant, which can be understood by structural modelling of the E1 and E2 conformations of the wild-type and mutant enzymes on the basis of the strategic location of the C terminus in relation to the third Na(+) binding site. The dramatic clinical presentation, together with the biochemical findings, provides both in vivo and in vitro evidence for a crucial role of the C terminus of the alpha-subunit in the function of the Na(+)/K(+)-ATPase and a key impact of Na(+) affinity in the pathophysiology of RDP.

The Rapid-onset Dystonia Parkinsonism Mutation D923N of the Na+,K+-ATPase α3 Isoform Disrupts Na+ Interaction at the Third Na+ Site

Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific α3-isoform of Na+,K+-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable ∼200-fold reduction of Na+ affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E1 form with intracellular Na+. This is the largest effect on Na+ affinity reported so far for any Na+,K+-ATPase mutant. D923N also affects the interaction with extracellular Na+ normally driving the E1P to E2P conformational transition backward. However, no impairment of K+ binding was observed for D923N, leading to the conclusion that Asp923 is specifically associated with the third Na+ site that is selective toward Na+. The crystal structure of the Na+,K+-ATPase in E2 form shows that Asp923 is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na+ ion. Structural modeling of the E1 form of Na+,K+-ATPase based on the Ca2+-ATPase crystal structure is consistent with the hypothesis that Asp923 contributes to a site binding the third Na+ ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na+ may be a general feature of the RDP disorder.

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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \([\mathrm{Rb}_{2}]{\cdot}E_{2}{\cdot}\mathrm{MgF}_{4}^{2-}\) \end{document} 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 Asp742 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 E2P 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 E2 to E1 conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K+/Rb+ site not only enhances E2P dephosphorylation but also stabilizes the E2 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 E2 state.

Importance of conserved Thr214 in domain A of the Na+,K+ -ATPase for stabilization of the phosphoryl transition state complex in E2P dephosphorylation.

Thr214 of the highly conserved214TGES sequence in domain A of the Na+,K+-ATPase was replaced with alanine, and the mutant was compared functionally with the previously characterized domain A mutant Gly263 → Ala. Thr214 → Ala displayed a conspicuous 150-fold reduction of the apparent vanadate affinity for inhibition of ATPase activity, which could not simply be explained by the observed shifts of the conformational equilibria in favor of E 1 andE 1P. The intrinsic vanadate affinity of theE 2 form and the effect on the apparent vanadate affinity of displacement of theE 1–E 2 equilibrium were determined in a phosphorylation assay that allows the enzyme-vanadate complex to be formed under equilibrium conditions. When theE 2 form prevailed, Thr214 → Ala retained a reduced vanadate affinity relative to wild type, whereas the affinity of Gly263 → Ala became wild type-like. Thus, mutation of Thr214 affected the intrinsic affinity ofE 2 for vanadate. Furthermore, Thr214 → Ala showed at least a 5-fold reducedE 2P dephosphorylation rate relative to wild type in the presence of saturating concentrations of K+ and Mg2+. Because vanadate is a phosphoryl transition state analog, it is proposed that defective binding of the phosphoryl transition state complex (transition state destabilization) causes the inability to catalyze E 2P dephosphorylation properly. By contrast, the phosphorylation site in theE 1 form was unaffected in Thr214→ Ala. Replacement of the glutamate, Glu216, of214TGES with alanine was incompatible with cell viability, indicating a very low transport activity or expression level. Our results support the hypothesis that domain A is isolated in the E 1 form, but contributes to make up the catalytic site in the E 2 and E 2P conformations.

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 α2 and α3 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 α 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 α2 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.

Functional Consequences of Alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal Part of Transmembrane Helix M3 of the Na+,K+-ATPase

Mutations Ile279 → Ala, Ile283 → Ala, Glu284 → Ala, His285 → Ala, His285 → Lys, His285 → Glu, Phe286 → Ala, and His288 → Ala in transmembrane helix M3 of the Na+,K+-ATPase were studied. Except for His285 → Ala, these mutations were compatible with cell viability, permitting analysis of their effects on the overall and partial reactions of the Na+,K+-transport cycle. In Ile279 → Ala and Ile283 → Ala, the E1 form accumulated, whereas in His285 → Lys and His285 → Glu, E1P accumulated. Phe286 → Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E2 and E2P, respectively, and showed a unique enhancement of the E1P → E2P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E1–E2 and E1P–E2P conformational changes. Because the E1–E2 and E1P–E2P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His285 furthermore increased the Na+-activated ATPase activity in the absence of K+ (“Na+-ATPase activity”). Ile279 → Ala, Ile283 → Ala, and His288 → Ala showed reduced Na+ affinity of the E1 form. The rate of Na+-activated phosphorylation from ATP was reduced in Ile279 → Ala and Ile283 → Ala, and these mutants showed evidence similar to Glu329 → Gln of destabilization of the Na+-occluded state.