Browse
Submit Data
Databases
API
Help

Dataset Information

36 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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 Mg²⁺. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na⁺,K⁺-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na⁺,K⁺-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

SUBMITTER: Ygberg S

PROVIDER: S-EPMC8637647 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase.

Project description:Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K(+). Studies with purified membranes revealed that CPZ reduced Na(+)-dependent phosphorylation by interference with Na(+) binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K(+). Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive (22)Na(+) influx into K(+)-loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na(+) cycle from an Na(+)/K(+) cycle in the pump. The Na(+) cycle possibly involves transport through the recently characterized Na(+)-specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na(+) efflux by modifying the transport stoichiometry of single pump units.

| S-EPMC2234217 | biostudies-literature

A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction.

Project description:Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in beta1 and alpha(I)-subunit genes of voltage-gated Na(+) channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R188W) [corrected] in the gene encoding the alpha-subunit of neuronal voltage-gated Na(+) channel type II (Na(v)1.2) in a patient with FS associated with afebrile seizures. The mutation R188W [corrected] occurring on Arg(187), a highly conserved residue among voltage-gated Na(+) channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNa(v)1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na(+) channel conductance was not affected. Prolonged residence in the open state of the R188W [corrected] mutant channel may augment Na(+) influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Na(v)1.2 in a human disease and propose the R188W [corrected] mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

| S-EPMC33477 | biostudies-literature

Mechanism of increased BK channel activation from a channel mutation that causes epilepsy.

Project description:Concerted depolarization and Ca(2+) rise during neuronal action potentials activate large-conductance Ca(2+)- and voltage-dependent K(+) (BK) channels, whose robust K(+) currents increase the rate of action potential repolarization. Gain-of-function BK channels in mouse knockout of the inhibitory beta 4 subunit and in a human mutation (alpha(D434G)) have been linked to epilepsy. Here, we investigate mechanisms underlying the gain-of-function effects of the equivalent mouse mutation (alpha(D369G)), its modulation by the beta 4 subunit, and potential consequences of the mutation on BK currents during action potentials. Kinetic analysis in the context of the Horrigan-Aldrich allosteric gating model revealed that changes in intrinsic and Ca(2+)-dependent gating largely account for the gain-of-function effects. D369G causes a greater than twofold increase in the closed-to-open equilibrium constant (6.6e(-7)-->1.65e(-6)) and an approximate twofold decrease in Ca(2+)-dissociation constants (closed channel: 11.3-->5.2 microM; open channel: 0.92-->0.54 microM). The beta 4 subunit inhibits mutant channels through a slowing of activation kinetics. In physiological recording solutions, we established the Ca(2+) dependence of current recruitment during action potential-shaped stimuli. D369G and beta 4 have opposing effects on BK current recruitment, where D369G reduces and beta 4 increases K(1/2) (K(1/2) microM: alpha(WT) 13.7, alpha(D369G) 6.3, alpha(WT)/beta 4 24.8, and alpha(D369G)/beta 4 15.0). Collectively, our results suggest that the D369G enhancement of intrinsic gating and Ca(2+) binding underlies greater contributions of BK current in the sharpening of action potentials for both alpha and alpha/beta 4 channels.

| S-EPMC2654085 | biostudies-literature

Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase.

Project description:Three Na+ sites are defined in the Na+-bound crystal structure of Na+, K+-ATPase. Sites I and II overlap with two K+ sites in the K+-bound structure, whereas site III is unique and Na+ specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na+ at site III, but does not contribute to K+ coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na+, K+-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Remarkably, Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane - in fact, mutations Q925A and Q925G increased the apparent Na+ affinity - but caused dramatic reductions of the binding of K+ as well as Na+ from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na+-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na+ at the extracellular side.

| S-EPMC6746705 | biostudies-literature

The Triad Na+ Activated Na+ Channel (Nax)-Salt Inducible KINASE (SIK) and (Na+ + K+)-ATPase: Targeting the Villains to Treat Salt Resistant and Sensitive Hypertension.

Project description:The Na+-activated Na+ channel (Nax) and salt-inducible kinase (SIK) are stimulated by increases in local Na+ concentration, affecting (Na+ + K+)-ATPase activity. To test the hypothesis that the triad Nax/SIK/(Na+ + K+)-ATPase contributes to kidney injury and salt-sensitive hypertension (HTN), uninephrectomized male Wistar rats (200 g; n = 20) were randomly divided into 4 groups based on a salt diet (normal salt diet; NSD-0.5% NaCl-or high-salt diet; HSD-4% NaCl) and subcutaneous administration of saline (0.9% NaCl) or deoxycorticosterone acetate (DOCA, 8 mg/kg), as follows: Control (CTRL), CTRL-Salt, DOCA, and DOCA-Salt, respectively. After 28 days, the following were measured: kidney function, blood pressure, (Na+ + K+)-ATPase and SIK1 kidney activities, and Nax and SIK1 renal expression levels. SIK isoforms in kidneys of CTRL rats were present in the glomerulus and tubular epithelia; they were not altered by HSD and/or HTN. CTRL-Salt rats remained normotensive but presented slight kidney function decay. HSD rats displayed augmentation of the Nax/SIK/(Na+ + K+)-ATPase pathway. HTN, kidney injury, and kidney function decay were present in all DOCA rats; these were aggravated by HSD. DOCA rats presented unaltered (Na+ + K+)-ATPase activity, diminished total SIK activity, and augmented SIK1 and Nax content in the kidney cortex. DOCA-Salt rats expressed SIK1 activity and downregulation in (Na+ + K+)-ATPase activity in the kidney cortex despite augmented Nax content. The data of this study indicate that the (Na+ + K+)-ATPase activity response to SIK is attenuated in rats under HSD, independent of HTN, as a mechanism contributing to kidney injury and salt-sensitive HTN.

| S-EPMC10178781 | biostudies-literature

Oxidative stress induced by the anti-cancer agents, plumbagin, and atovaquone, inhibits ion transport through Na+/K+-ATPase.

Project description:Oxidative stress inhibits Na+/K+-ATPase (NKA), the ion channel that maintains membrane potential. Here, we investigate the role of oxidative stress-mediated by plumbagin and atovaquone in the inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit the proliferation of three human (OVCAR-3, SKOV-3, and TYKNu) and one mouse (ID8) ovarian cancer cell lines. The oxygen radical scavenger, N-acetylcysteine (NAC), attenuates the chemotoxicity of plumbagin and atovaquone. Whole-cell patch clamping demonstrates that plumbagin and atovaquone inhibit outward and the inward current flowing through NKA in SKOV-3 and OVCAR-3. Although both drugs decrease cellular ATP; providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either drug significantly decreases the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA, resulting in the inability to maintain ion transport. Therefore, when evaluating compounds that induce oxidative stress, it is important to consider the contribution of NKA inhibition to their cytotoxic effects on tumor cells.

| S-EPMC7659016 | biostudies-literature

β subunit affects Na+ and K+ affinities of Na+/K+-ATPase: Na+ and K+ affinities of a hybrid Na+/K+-ATPase composed of insect α and mammalian β subunits.

Project description:The affinity for K+ of silkworm Na+/K+-ATPase, which is composed of α and β subunits, is remarkably lower than that of mammalian Na+/K+-ATPase, with a slightly higher affinity for Na+. Because the α subunit had more than 70% identity to the mammalian α subunit in the amino acid sequence, whereas the β subunit, a glycosylated protein, had less than 30% identity to the mammalian β subunit, it was suggested that the β subunit was involved in the affinities for Na+ and K+ of Na+/K+-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm β subunit with the mammalian β subunit affected the affinities for Na+ and K+ of Na+/K+-ATPase. Cloned silkworm α and cloned rat β1 were co-expressed in BM-N cells, a cultured silkworm ovary-derived cell lacking endogenous Na+/K+-ATPase, to construct a hybrid Na+/K+-ATPase, in which the silkworm β subunit was replaced with the rat β1 subunit. The hybrid Na+/K+-ATPase increased the affinity for K+ by 4.1-fold and for Na+ by 0.65-fold compared to the wild-type one. Deglycosylation of the silkworm β subunit did not affect the K+ affinity. These results support the involvement of the β subunit in the Na+ and K+ affinities of Na+/K+-ATPase.

| S-EPMC9483571 | biostudies-literature

Na+/vacancy disordering promises high-rate Na-ion batteries.

Project description:As one of the most fascinating cathode candidates for Na-ion batteries (NIBs), P2-type Na layered oxides usually exhibit various single-phase domains accompanied by different Na+/vacancy-ordered superstructures, depending on the Na concentration when explored in a limited electrochemical window. Therefore, their Na+ kinetics and cycling stability at high rates are subjected to these superstructures, incurring obvious voltage plateaus in the electrochemical profiles and insufficient battery performance as cathode materials for NIBs. We show that this problem can be effectively diminished by reasonable structure modulation to construct a completely disordered arrangement of Na-vacancy within Na layers. The combined analysis of scanning transmission electron microscopy, ex situ x-ray absorption spectroscopy, and operando x-ray diffraction experiments, coupled with density functional theory calculations, reveals that Na+/vacancy disordering between the transition metal oxide slabs ensures both fast Na mobility (10-10 to 10-9 cm2 s-1) and a low Na diffusion barrier (170 meV) in P2-type compounds. As a consequence, the designed P2-Na2/3Ni1/3Mn1/3Ti1/3O2 displays extra-long cycle life (83.9% capacity retention after 500 cycles at 1 C) and unprecedented rate capability (77.5% of the initial capacity at a high rate of 20 C). These findings open up a new route to precisely design high-rate cathode materials for rechargeable NIBs.

| S-EPMC5844706 | biostudies-literature

Na Channel ? Subunits: Overachievers of the Ion Channel Family.

Project description:Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming ? subunit and one or more ? subunits. There are five mammalian ? subunits in total: ?1, ?1B, ?2, ?3, and ?4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, ?1B, the ? subunits are type I topology transmembrane proteins. In contrast, ?1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC ? subunits are multifunctional. While they do not form the ion channel pore, ? subunits alter gating, voltage-dependence, and kinetics of VGSC? subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, ? subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. ? subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of ? subunits is ?1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and ?-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. ? subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. ? subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some ? subunit functions may operate independently of ? subunits. Thus, ? subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.

| S-EPMC3181431 | biostudies-literature

The Na+,K+-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state.

Project description:The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2-BeFx). E2-BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9-7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2-BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.

| S-EPMC9485054 | biostudies-literature