Vivien Rodacker Schack

Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na+/K+ ATPase α subunit) and ATP2B3 (encoding a Ca2+ ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.

Mutation I810N in the α3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na+,K+-ATPase α3 isoform inactive. Total Na+,K+-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na+,K+-ATPase α3 by transgenesis, which also rescued Na+,K+-ATPase activity. Our findings reveal the functional significance of the Na+,K+-ATPase α3 isoform in the control of epileptiform activity and seizure behavior.

Somatic ATP1A1, ATP2B3, and KCNJ5 Mutations in Aldosterone-Producing Adenomas

Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein–activated inward rectifier K+ channel 4, GIRK4, account for ≈40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na+/K+-ATPase 1 and Ca2+-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na+/K+-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na+ activation of phosphorylation and K+ inhibition of phosphorylation that indicate decreased Na+ and K+ binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na+/K+-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.

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).

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.

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.

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.

Inhibition of Phosphorylation of Na+,K+-ATPase by Mutations Causing Familial Hemiplegic Migraine

Background: Familial hemiplegic migraine type II (FHM2) is caused by mutations in the Na+,K+-ATPase α2-isoform. Results: Several FHM2 mutations inhibit phosphorylation or dephosphorylation. Conclusion: These mutations cause FHM2 by local and long range effects on the catalytic site and not by reducing the affinity for external K+. Significance: Insights into the pathophysiological mechanism of FHM2 and the molecular mechanism of the Na+,K+-ATPase have been obtained. 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 E2P 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 E1P to E2P transition.

Neurological disease mutations of α3 Na(+),K(+)-ATPase: Structural and functional perspectives and rescue of compromised function.

Na+,K+-ATPase creates transmembrane ion gradients crucial to the function of the central nervous system. The α-subunit of Na+,K+-ATPase exists as four isoforms (α1-α4). Several neurological phenotypes derive from α3 mutations. The effects of some of these mutations on Na+,K+-ATPase function have been studied in vitro. Here we discuss the α3 disease mutations as well as information derived from studies of corresponding mutations of α1 in the light of the high-resolution crystal structures of the Na+,K+-ATPase. A high proportion of the α3 disease mutations occur in the transmembrane sector and nearby regions essential to Na+ and K+ binding. In several cases the compromised function can be traced to disturbance of the Na+ specific binding site III. Recently, a secondary mutation was found to rescue the defective Na+ binding caused by a disease mutation. A perspective is that it may be possible to develop an efficient pharmaceutical mimicking the rescuing effect.