A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase.

Association of Familial Hyperkalemia and Hypertension with Proximal Renal Tubular Acidosis and Epileptic Seizures.

Introduction: Familial hyperkalemic hypertension (FHHt) is an inherited disease characterized by hyperkalemia, hypertension, and hyperchloremic acidosis (HCA). The primary defect is a hyperactive sodium chloride co-transporter, expressed in the renal distal tubule. FHHt is caused by mutation in either WNK1, WNK4, KLHL3, or Cul3.

A missense mutation converts the Na,K-ATPase into an ion channel and causes therapy-resistant epilepsy.

The ion pump Na,K-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na,K-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg.

Revisiting the binding kinetics and inhibitory potency of cardiac glycosides on Na,K-ATPase (α1β1): Methodological considerations.

Introduction: Ouabain and digoxin are classical inhibitors of the Na,K-ATPase. In addition to their conventional uses as therapeutic agents or experimental tools there is renewed interest due to evidence suggesting they could be endogenous hormones. Somewhat surprisingly, different publications show large discrepancies in potency for inhibiting Na,K-ATPase activity (IC), particularly for the slow binding inhibitors, ouabain and digoxin.

Renal Mg handling, FXYD2 and the central role of the Na,K-ATPase.

This article examines the central role of Na,K-ATPase (α1β1FXYD2) in renal Mg handling, especially in distal convoluted tubule (DCT), the segment responsible for final regulation of Mg balance. By considering effects of Na,K-ATPase on intracellular Na and K concentrations, and driving forces for Mg transport, we propose a consistent rationale explaining basal Mg reabsorption in DCT and altered Mg reabsorption in some human diseases. FXYD2 (γ subunit) is a regulatory subunit that adapts functional properties of Na,K-ATPase to cellular requirements.

Hypermagnesuria in Humans Following Acute Intravenous Administration of Digoxin.

Background: Hypomagnesemia is a known predisposing condition for the appearance of digitalis toxicity. The detection of a genetic form of Mg urinary wasting with hypomagnesemia being caused by a mutation in the γ subunit (FXYD2) of the Na,K-ATPase, the pharmacological target of Digoxin, prompted us to investigate whether Digoxin administration increases urinary Mg excretion.

Methods: Two groups of subjects, with rapid atrial fibrillation, received intravenous Digoxin (n = 9) or verapamil (n = 8), for heart rate control.

Specific phospholipid binding to Na,K-ATPase at two distinct sites.

Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity.

Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.

The Na,K-ATPase α subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca, whereas α has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α in the heart is related to selective assembly with a particular one of the three β isoforms.

Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

Much evidence points to a role of Na,K-ATPase in ouabain-dependent signal transduction. Based on experiments with different cell lines and native tissue membranes, a current hypothesis postulates direct interactions between the Na,K-ATPase and Src kinase (non-receptor tyrosine kinase). Na,K-ATPase is proposed to bind Src kinase and inhibit its activity, whereas ouabain, the specific Na,K-ATPase inhibitor, binds and stabilizes the E2 conformation, thus exposing the Src kinase domain and its active site Tyr-418 for activation.

Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure.

The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1β1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2β3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2β3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO4 oxidation of the third digitoxose and reductive amination with various R-NH2 substituents and identified derivatives with significant selectivity for human α2β1 over α1β1 (up to 7.

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase.

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms.

Cardiac glycosides induced toxicity in human cells expressing α1-, α2-, or α3-isoforms of Na-K-ATPase.

The Na+-K+-ATPase is specifically inhibited by cardiac glycosides, some of which may also function as endogenous mammalian hormones. Previous studies using Xenopus oocytes, yeast cells, or purified isoforms demonstrated that affinities of various cardiac glycosides for three isoforms of the Na+-K+-ATPase (α1-α3β1) may differ, a finding with potential clinical implication. The present study investigates isoform selectivity and effects of cardiac glycosides on cultured mammalian cells under more physiological conditions.

Stimulation, inhibition, or stabilization of Na,K-ATPase caused by specific lipid interactions at distinct sites.

The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase.

Digoxin derivatives with enhanced selectivity for the α2 isoform of Na,K-ATPase: effects on intraocular pressure in rabbits.

In the ciliary epithelium of the eye, the pigmented cells express the α1β1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2β3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea.

Ouabain-induced internalization and lysosomal degradation of the Na+/K+-ATPase.

Internalization of the Na(+)/K(+)-ATPase (the Na(+) pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors.

Neutral phospholipids stimulate Na,K-ATPase activity: a specific lipid-protein interaction.

Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein.

Stabilization of the α2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket.

The α2 isoform of Na,K-ATPase plays a crucial role in Ca(2+) handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1β1, α2β1, and α3β1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 ≫ α3 > α1).

Phospholemman (FXYD1) raises the affinity of the human α1β1 isoform of Na,K-ATPase for Na ions.

The human α(1)/His(10)-β(1) isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside, and purified by metal chelate chromatography. The α(1)β(1) complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α(1)/His(10)-β(1) complex and stabilizes it in an active mode.

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions.

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified.

Selectivity of digitalis glycosides for isoforms of human Na,K-ATPase.

There are four isoforms of the alpha subunit (alpha1-4) and three isoforms of the beta subunit (beta1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. alpha2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An alpha2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of alpha2.

Neutralization of the charge on Asp 369 of Na+,K+-ATPase triggers E1 <--> E2 conformational changes.

This work investigates the role of charge of the phosphorylated aspartate, Asp(369), of Na(+),K(+)-ATPase on E(1) <--> E(2) conformational changes. Wild type (porcine alpha(1)/His(10)-beta(1)), D369N/D369A/D369E, and T212A mutants were expressed in Pichia pastoris, labeled with fluorescein 5'-isothiocyanate (FITC), and purified. Conformational changes of wild type and mutant proteins were analyzed using fluorescein fluorescence (Karlish, S.

A C-terminal lobe of the beta subunit of Na,K-ATPase and H,K-ATPase resembles cell adhesion molecules.

The beta subunit of Na,K-ATPase is required for stabilization and maturation of the catalytic alpha subunits and is also involved in cell adhesion and establishing epithelial cell polarity. However, the mechanism of cell adhesion effects and protein partners of beta are unknown. We have applied fold recognition methods to predict that a C-terminal domain of the beta subunits of Na,K-ATPase and H,K-ATPase has an immunoglobulin-like fold, which resembles cell adhesion molecules.

Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1.

Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state.