Jan Joep H. H. M. De Pont

Dominant isolated renal magnesium loss is caused by misrouting of the Na(+),K(+)-ATPase gamma-subunit.

Primary hypomagnesaemia is composed of a heterogeneous group of disorders characterized by renal or intestinal Mg2+ wasting, often associated with disturbances in Ca2+ excretion. We identified a putative dominant-negative mutation in the gene encoding the Na+,K+-ATPase γ-subunit (FXYD2), leading to defective routing of the protein in a family with dominant renal hypomagnesaemia.

Dominant Isolated Renal Magnesium Loss Is Caused by Misrouting of the Na+,K+‐ATPase γ‐Subunit

Abstract: Hereditary primary hypomagnesemia comprises a clinically and genetically heterogeneous group of disorders in which hypomagnesemia is due to either renal or intestinal Mg2+ wasting. These disorders share the general symptoms of hypomagnesemia, tetany and epileptiformic convulsions, and often include secondary or associated disturbances in calcium excretion. In a large Dutch family with autosomal dominant renal hypomagnesemia, associated with hypocalciuria, we mapped the disease locus to a 5.6‐cM region on chromosome 11q23. After candidate screening, we identified a heterozygous mutation in the FXYD2 gene, encoding the Na+,K+‐ATPase γ‐subunit, cosegregating with the patients of this family, which was not found in 132 control chromosomes. The mutation leads to a G41R substitution, introducing a charged amino acid residue in the predicted transmembrane region of the γ‐subunit protein. Expression studies in insect Sf9 and COS‐1 cells showed that the mutant γ‐subunit protein was incorrectly routed and accumulated in perinuclear structures. In addition to disturbed routing of the G41R mutant, Western blot analysis of Xenopus oocytes expressing wild‐type or mutant γ‐subunit showed mutant γ‐subunit lacking a posttranslational modification. Finally, we investigated two individuals lacking one copy of the FXYD2 gene and found their serum Mg2+ levels to be within the normal range. We conclude that the arrest of mutant γ‐subunit in distinct intracellular structures is associated with aberrant posttranslational processing and that the G41R mutation causes dominant renal hypomagnesemia associated with hypocalciuria through a dominant negative mechanism.

Functional expression of gastric H,K-ATPase using the baculovirus expression system

A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the α‐ as well as the β‐subunit of the gastric H.K‐ATPase. Sf9 cells infected with this virus exhibited a K+‐ and SCH 28080‐sensitive ATP‐dependent phosphorylation capacity in purified Sf9 membranes similar to Natlve H,K‐ATPase. This activity was not present in control membranes containing only one of the two H,K‐ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H.K‐ATPase.

Constitutive activation of gastric H+,K+‐ATPase by a single mutation

In the reaction cycle of P‐type ATPases, an acid‐stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane‐bound enzymes. In some of these ATPases, such as Na+,K+‐ATPase and gastric H+,K+‐ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+‐ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the α‐subunit, have a high SCH 28080‐sensitive, but K+‐insensitive ATPase activity. This high activity is caused by an increased ‘spontaneous’ rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K+, but K+ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.

Effect of free fatty acids and detergents on H,K-ATPase. The steady-state ATP phosphorylation level and the orientation of the enzyme in membrane preparations

The effects of detergents and free fatty acids on the K(+)-activated ATPase activity and on the steady-state phosphorylation level of pig gastric H,K-ATPase were studied. Unsaturated free fatty acids inhibited the K(+)-activated ATPase activity, due to inactivation of the enzyme (long-term effects) and to a decrease in the K(+)-sensitive dephosphorylation rate (short-term effects). The degree of inhibition depended on the reaction conditions: the protein concentration, the temperature and the ligands used. No effect was observed when saturated- or methylated unsaturated fatty acids were tested. Free fatty acids and the detergent C12E8 increased the steady-state ATP phosphorylation level, indicating the presence of vesicular structures in the H,K-ATPase preparations. At higher concentrations these compounds inactivated H,K-ATPase, which was measured as a decrease in phosphorylation capacity. By combining the data from the ATP phosphorylation level in the absence and presence of C12E8 (without inactivation) and the data from the K(+)-activated ATPase activity with and without ionophore the tightness of vesicular preparations and the orientation of H,K-ATPase was determined. A rather simple method for the isolation of H,K-ATPase is reported, which yields highly purified H,K-ATPase preparations with a ATP phosphorylation capacity of 3.9 nmol P per mg protein or 0.57 mol P per mol alpha beta protomer. This number suggests that each alpha-subunit H,K-ATPase can be phosphorylated at the same time.

The β-Subunits of Na+,K+-ATPase and Gastric H+,K+-ATPase Have a High Preference for Their Own α-Subunit and Affect the K+ Affinity of These Enzymes

The α- and β-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the α-subunits resulted in coprecipitation of the accompanying β-subunit independent of the type of β-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the β-subunit of H+,K+-ATPase (NaKαHKβ) showed an ATPase activity, which was only 12 ± 4% of the activity of the Na+,K+-ATPase with its own β-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the β-subunit of Na+,K+-ATPase (HKαNaKβ) showed an ATPase activity which was 9 ± 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKαHKβ was decreased, while the apparent K+ affinity of the opposite hybrid HKαNaKβ was increased. The hybrid NaKαHKβ could be phosphorylated by ATP to a level of 21 ± 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKαβ and NaKαHKβ of 8800 ± 310 min−1 and 4800 ± 160 min−1, respectively. Measurements of phosphorylation of the HKαNaKβ and HKαβ enzymes are consistent with a higher turnover of the former. These findings suggest a role of the β-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own β-subunit, they can function with the β-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.

Conversion of the low affinity ouabain-binding site of non-gastric H,K-ATPase into a high affinity binding site by substitution of only five amino acids.

P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349–32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase β1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document}-stimulated ATPase activity in membranes of transfected Sf9 cells.

Gastric H+/K+-ATPase

The gastric H+/K+-ATPase is an enzyme located in the tubu-lovesicular system and the apical membrane of the parietal cell and plays a crucial role in the process of gastric acid secretion. The enzyme catalyses an electroneutral exchange of H+ versus K+. It consists of a catalytic α-subunit and a heavily glycosylated β-subunit, both of which are rather homologous to their counterparts in Na+/K+-ATPase. In this review, molecular and genetic aspects of this enzyme are reviewed with reference to similar enzymes in other tissues.

Transport ratios of reconstituted (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol vesicles by means of a freeze-thaw-sonication procedure. The passive and active transport mediated by these vesicles were measured (Skrabanja, A.T.P., Asty, P., Soumarmon, A., De Pont, J.J.H.H.M. and Lewin, M.J.M. (1986) Biochim. Biophys. Acta 860, 131-136). To determine real initial velocities, the proteoliposomes were separated from non-incorporated enzyme, by means of centrifugation on a sucrose gradient. The purified proteoliposomes were used to measure active H+ and Rb+ transport, giving at room-temperature velocities of 46.3 and 42.5 mumol per mg per h, respectively. A transport ratio of two cations per ATP hydrolyzed was also measured. These figures indicate that the enzyme catalyzes an electroneutral H+-Rb+ exchange.

Phosphorylation of H+/K+-ATPase by inorganic phosphate. The role of K+ and SCH 28080

The effects of K+ on the phosphorylation of H+/K(+)-ATPase with inorganic phosphate were studied using H+/K(+)-ATPase purified from porcine gastric mucosa. The phosphoenzyme formed by phosphorylation with Pi was identical with the phosphoenzyme formed with ATP. The maximal phosphorylation level obtained with Pi was equal to that obtained with ATP. The Pi phosphorylation reaction of H+/K(+)-ATPase was, like that of Na+/K(+)-ATPase, a relatively slow reaction. The rates of phosphorylation and dephosphorylation were both increased by low concentrations of K+, which resulted in hardly any effect on the phosphorylation level. A decrease of the steady-state phosphorylation level was caused by higher concentrations of K+ in a noncompetitive manner, whereas no further increase in the dephosphorylation rate was observed. The decreasing effect was caused by a slow binding of K+ to the enzyme. All above-mentioned K+ effects were abolished by the specific H+/K(+)-ATPase inhibitor SCH 28080 (2-methyl-8-[phenyl-methoxy]imidazo-[1-2-a]pyrine-3-acetonitrile). Additionally, SCH 28080 caused a 2-fold increase in the affinity of H+/K(+)-ATPase for Pi. A model for the reaction cycle of H+/K(+)-ATPase fitting the data is postulated.