Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na þ , K þ-ATPase a1 subunit (ATP1A1). Functional characterization of mutant Na þ , K þ-ATPase a1 subunits in heterologous expression systems revealed not only a loss of Na þ , K þ-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the a1 isoform of the Na þ , K þ-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity.
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-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.
Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific ␣3-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 ϳ200-fold reduction of Na ؉ affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E 1 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 E 1 P to E 2 P conformational transition backward. However, no impairment of K ؉ binding was observed for D923N, leading to the conclusion that Asp 923 is specifically associated with the third Na ؉ site that is selective toward Na ؉ . The crystal structure of the Na ؉ ,K ؉ -ATPase in E 2 form shows that Asp 923 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 E 1 form of Na ؉ ,K ؉ -ATPase based on the Ca 2؉ -ATPase crystal structure is consistent with the hypothesis that Asp 923 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.
Synaptic activity results in transient elevations in extracellular K , clearance of which is critical for sustained function of the nervous system. The K clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na /K -ATPase. The Na /K -ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na /K -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na /K -ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2 mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K dynamics.
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