Potassium translocation by the Na+/K+ pump is voltage insensitive.

The voltage dependence of steady and transient changes in Na+/K+ pump current, in response to step changes in membrane potential, was investigated in guinea pig ventricular myocytes voltage clamped and internally dialyzed under experimental conditions designed to support four separate modes of Na+/K+ pump activity. Voltage jumps elicited transient pump currents when the pump cycle was running forward or backward, or when pumps were limited to Na+ translocation, but not when they were made to carry out K+/K+ exchange. This result indicates that K+ translocation involves no net charge movement across the membrane field and is therefore voltage insensitive. The transient pump currents seen during Na+/K+ transport demonstrate that both forward and reverse pump cycles are rate limited not by the voltage-dependent step but by a voltage-independent step, probably K+ translocation. These findings severely constrain kinetic models of Na+/K+ pump activity.     

K+-induced dilation of hamster cremasteric arterioles involves both the Na+/K+-ATPase and inward-rectifier K+ channels

OBJECTIVE: The mechanism by which elevated extracellular potassium ion concentration ([K+]o) causes dilation of skeletal muscle arterioles was evaluated. METHODS: Arterioles (n = 111) were hand-dissected from hamster cremaster muscles, cannulated with glass micropipettes and pressurized to 80 cm H2O for in vitro study. The vessels were superfused with physiological salt solution containing 5 mM KCl, which could be rapidly switched to test solutions containing elevated [K+]o and/or inhibitors. The authors measured arteriolar diameter with a computer-based diameter tracking system, vascular smooth muscle cell membrane potential with sharp micropipettes filled with 200 mM KCl, and changes in intracellular Ca2+ concentration ([Ca2+]i) with Fura 2. Membrane currents and potentials also were measured in enzymatically isolated arteriolar muscle cells using patch clamp techniques. The role played by inward rectifier K+ (KIR) channels was tested using Ba2+ as an inhibitor. Ouabain and substitution of extracellular Na+ with Li+ were used to examine the function of the Na+/K+ ATPase. RESULTS: Elevation of [K+]o from 5 mM up to 20 mM caused transient dilation of isolated arterioles (27 +/- 1 microm peak dilation when [K+]o was elevated from 5 to 20 mM, n = 105, p <.05). This dilation was preceded by transient membrane hyperpolarization (10 +/-1 mV, n = 23, p <.05) and by a fall in [Ca2+]i as indexed by a decrease in the Fura 2 fluorescence ratio of 22 +/- 5% (n = 4, p <.05). Ba(2+) (50 or 100 microM) attenuated the peak dilation (40 +/- 8% inhibition, n = 22) and hyperpolarization (31 +/- 12% inhibition, n = 7, p <.05) and decreased the duration of responses by 37 +/-11% (n = 20, p < 0.05). Both ouabain (1 mM or 100 microM) and replacement of Na+ with Li+ essentially abolished both the hyperpolarization and vasodilation. CONCLUSIONS: Elevated [K+]o causes transient vasodilation of skeletal muscle arterioles that appears to be an intrinsic property of the arterioles. The results suggest that K+-induced dilation involves activation of both the Na+/K+ ATPase and KIR channels, leading to membrane hyperpolarization, a fall in [Ca2+]i, and culminating in vasodilation. The Na+/K+ ATPase appears to play the major role and is largely responsible for the transient nature of the response to elevated [K+]o, whereas KIR channels primarily affect the duration and kinetics of the response.     

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase alpha2-isoform in heart failure

AIMS: The Na+/K+-ATPase (NKA) alpha2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca(+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA alpha2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. METHODS AND RESULTS: NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (I NKA and I NCX) were measured and alpha2-isoform current (I NKA,alpha2) was separated from total I NKA using 0.3 microM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of alpha2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA alpha1- and alpha2-isoforms. I NKA,alpha2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When I NKA,alpha2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of I NKA led to a decrease in I NCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the alpha2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the alpha2-isoform is necessary to modulate local [Na+]i close to NCX. CONCLUSION: Downregulation of the alpha2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.     

The functional effect of adenoviral Na+/Ca2+ exchanger overexpression in rabbit myocytes depends on the activity of the Na+/K+-ATPase

OBJECTIVES: The functional consequences of Na+/Ca2+ exchanger (NCX) overexpression in heart failure have been controversially discussed. NCX function strongly depends on intracellular sodium which has been shown to be increased in heart failure. METHODS AND RESULTS: We investigated the Na+/K+-ATPase (NKA) inhibitor ouabain (0.5-16 micromol/l) in electrically stimulated, isotonically contracting adult rabbit cardiocytes overexpressing NCX after adenoviral gene transfer (Ad-NCX-GFP, 48 h culture time). Myocytes transfected with adenovirus encoding for green fluorescent protein (Ad-GFP) served as a control. Contractions were analyzed by video-edge detection. In the Ad-NCX-GFP group, the maximum inotropic response was significantly reduced by 50.7% (P<0.05). This was a result of an enhanced susceptibility to contracture after exposure to the drug (median concentration (25-75%): 4 (4-8) vs. 8 (6-16) micromol/l, P<0.05). When analyzing relaxation before contracture, the maximum relaxation velocity was reduced (0.15+/-0.04 vs. 0.27+/-0.04 microm/s, P<0.05) and the time from peak shortening to 90% of relaxation was increased (298+/-39 vs. 185+/-15 ms, P<0.05). No differences in systolic and diastolic parameters were observed with the Na+ channel modulator BDF9198 (1 micromol/l). CONCLUSIONS: Inhibition of NKA by ouabain induces a combined diastolic and systolic dysfunction in NCX overexpressing rabbit myocytes. This may be the consequence of cytoplasmic Ca2+ overload due to inhibition of forward mode or induction of reverse mode Na+/Ca2+ exchange. In end-stage failing human myocardium and during digitalis treatment this mechanism may be of major importance.     

Inhibition of Rat Na+/K+-ATPase Isoforms by Endogenous Digitalis Extracts from Neonatal Human Plasma

An unique endogeneous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (α₁, α₂and α₃) of the alpha subunits of Na⁺/K⁺-ATPase in rat

EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral α₃isoform and a steep dose-response curve with the same apparent potency for rat α₂and α₁isoforms of high (10^-7M) and low affinity (3 × 10^-5M) for ouabain. These results indicate that the Na⁺/K⁺-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain     

Ischemic preconditioning prevents calpain-mediated impairment of Na+/K+-ATPase activity during early reperfusion

OBJECTIVES: We previously demonstrated that ischemic preconditioning (IPC) attenuates calpain activation during reperfusion. Herein, we tested the hypothesis that enhancement of Na+/K+-ATPase activity during early reperfusion as a result of calpain inhibition is involved in the protection afforded by myocardial IPC. METHODS: Intracellular Na+ concentration ([Na+]i) measured using 23Na-magnetic resonance spectroscopy, Na+/K+-ATPase activity, detachment of Na+/K+-ATPase alpha subunits from the membrane cytoskeleton, degradation of fodrin and ankyrin, and calpain activation were analysed in isolated rat hearts reperfused after 60 min of ischemia with or without previous IPC and different treatments aimed to mimic or blunt the effects of IPC. RESULTS: In non-treated hearts subjected to ischemia (control hearts), reperfusion for 5 min severely reduced Na+/K+-ATPase activity and dissociated alpha1 and alpha2 subunits of Na+/K+-ATPase from the membrane-cytoskeleton complex in parallel with proteolysis of alpha-fodrin and ankyrin-B and calpain activation. IPC accelerated the recovery of [Na+]i, increased Na+/K+-ATPase activity, and prevented dissociation of Na+/K+-ATPase from the membrane-cytoskeleton complex. IPC also prevented alpha-fodrin and ankyrin-B loss and calpain activation, effects that were associated with attenuated lactate dehydrogenase (LDH) release and infarct size and improved contractile recovery. These effects of IPC were reproduced by perfusing the hearts with the calpain inhibitor MDL-28170 and by transient stimulation of cAMP-dependent protein kinase (PKA) with CPT-cAMP, and they were reverted by perfusing with the PKA inhibitor H89. CONCLUSION: The results of the present study are consistent with the hypothesis that enhanced recovery of Na+/K+-ATPase activity during reperfusion as a result of attenuated calpain-mediated detachment of the protein from the membrane-cytoskeleton complex contributes to the protection afforded by IPC.     

Mouse Na+/K+-ATPase beta1-subunit has a K+-dependent cell adhesion activity for beta-GlcNAc-terminating glycans

A 48-kDa beta-N-acetylglucosamine (GlcNAc)-binding protein was isolated from mouse brain by GlcNAc-agarose column chromatography. The N-terminal amino acid residues showed the protein to be a mouse Na(+)/K(+)-ATPase beta1-subunit. When the recombinant FLAG-beta1-subunit expressed in Sf-9 cells was applied to a GlcNAc-agarose column, only the glycosylated 38- and 40-kDa proteins bound to the column. In the absence of KCl, little of the proteins bound to a GlcNAc-agarose column, but the 38- and 40-kDa proteins bound in the presence of KCl at concentrations above 1 mM. Immunohistochemical study showed that the beta1-subunit and GlcNAc-terminating oligosaccharides are at the cell contact sites. Inclusion of anti-beta1-subunit antibody or chitobiose in cell aggregation assays using mouse neural cells resulted in inhibition of cell aggregation. These results indicate that the Na(+)/K(+)-ATPase beta1-subunit is a potassium-dependent lectin that binds to GlcNAc-terminating oligosaccharides: it may be involved in neural cell interactions.     

The Na+/K+-ATPase alpha2-isoform regulates cardiac contractility in rat cardiomyocytes

OBJECTIVE: The presence of both alpha1- and alpha2-isoforms of the Na+/K+-ATPase (NKA) in cardiomyocytes indicates different functions. We hypothesized that preferential localization of the alpha2-isoform to the t-tubules, locally controlling the Na+/Ca2+-exchanger (NCX), underlies a specific role in Ca2+ handling. METHODS: We studied NKA isoform distribution in isolated cardiomyocytes from Wistar rats using immunocytochemistry. NKA pump and NCX currents (I(pump) and I(NCX)) were measured in control and detubulated cardiomyocytes. Intracellular Na+ concentration [Na+]i was assessed with the fluorescent dye SBFI. RESULTS: The alpha2-isoform abundance was higher in the t-tubules than in the surface sarcolemma. We established that 0.3 microM ouabain specifically blocked the alpha2-isoform in isolated rat cardiomyocytes. This low concentration blocked 10.7+/-0.6% of I(pump) in control, but only 6.0+/-0.5% in detubulated cardiomyocytes. Moreover, measured and calculated alpha1-specific and alpha2-specific I(pump) in control (547+/-29 pA and 66 pA, respectively) and in detubulated cells (495+/-30 pA and 31 pA, respectively) showed that 53% of the alpha2-isoform, but only 9.5% of the alpha1-isoform, were localized to the t-tubules. Despite the small abundance of the alpha2-isoform (approximately 11% of total NKA), selective inhibition of this isoform induced a 40% increase in contractility in field stimulated cardiomyocytes, but no increase in global [Na+]i. However, inhibition of the alpha2-isoform increased I(NCX) indicating local subsarcolemmal accumulation of Na+ near NCX. CONCLUSIONS: The alpha2-isoform of the NKA is functionally coupled to the NCX and can regulate Ca2+ handling without changing global [Na+]i.     

Na+,K+-ATP酶与脑缺血

Na+,K+-ATP酶的基本功能是维持真核细胞膜内外Na+ - K+电化学梯度平衡,后者为维持细胞渗透压、调节细胞体积和维持可兴奋细胞膜静息电位所必需.Na+,K+ -ATP酶活性的维持在神经元神经递质的摄取和Ca2+外流中起着重要作用.脑缺血后,Na+,K+ -ATP酶活性降低及功能异常参与缺血性脑损伤过程.缺血预处理通过维持缺血后Na+,K+ -ATP酶活性而诱导缺血耐受.强心甾类固醇和胞二磷胆碱可通过提高Na+,K+ -ATP酶活性对脑缺血发挥神经保护效应.     

Na+,K+-ATP酶与脑缺血

Na+,K+-ATP酶的基本功能是维持真核细胞膜内外Na+ - K+电化学梯度平衡,后者为维持细胞渗透压、调节细胞体积和维持可兴奋细胞膜静息电位所必需.Na+,K+ -ATP酶活性的维持在神经元神经递质的摄取和Ca2+外流中起着重要作用.脑缺血后,Na+,K+ -ATP酶活性降低及功能异常参与缺血性脑损伤过程.缺血预处理通过维持缺血后Na+,K+ -ATP酶活性而诱导缺血耐受.强心甾类固醇和胞二磷胆碱可通过提高Na+,K+ -ATP酶活性对脑缺血发挥神经保护效应.     

Na+,K+-ATP酶与脑缺血

Na+,K+-ATP酶的基本功能是维持真核细胞膜内外Na+ - K+电化学梯度平衡,后者为维持细胞渗透压、调节细胞体积和维持可兴奋细胞膜静息电位所必需.Na+,K+ -ATP酶活性的维持在神经元神经递质的摄取和Ca2+外流中起着重要作用.脑缺血后,Na+,K+ -ATP酶活性降低及功能异常参与缺血性脑损伤过程.缺血预处理通过维持缺血后Na+,K+ -ATP酶活性而诱导缺血耐受.强心甾类固醇和胞二磷胆碱可通过提高Na+,K+ -ATP酶活性对脑缺血发挥神经保护效应.     

Na+/K+-ATP酶ATP1A2基因突变与偏头痛

偏头痛是一种常见的反复发作性神经疾病。病因涉及遗传和环境因素,其发病机制尚未完全阐明。家族性偏瘫型偏头痛(familial hemiplegic migraine,FHM)中Na /K ATP酶α2亚基ATP1A2基因突变的发现,是偏头痛遗传学研究的重大突破。目前已鉴定出25种ATP1A2基因突变,且绝大部分为错义突变,这些突变或者引起Na /K -ATP酶α2亚基单个等位基因的功能丧失,或者引起钠钾泵的动力学改变。不过,ATP1A2基因与普通偏头痛的关系尚不明确,有待进一步研究。     

Interaction between Na+/K+-pump and Na+/Ca2+-exchanger modulates intercellular communication

Ouabain, a specific inhibitor of the Na(+)/K(+)-pump, has previously been shown to interfere with intercellular communication. Here we test the hypothesis that the communication between vascular smooth muscle cells is regulated through an interaction between the Na(+)/K(+)-pump and the Na(+)/Ca(2+)-exchanger leading to an increase in the intracellular calcium concentration ([Ca(2+)](i)) in discrete areas near the plasma membrane. [Ca(2+)](i) in smooth muscle cells was imaged in cultured rat aortic smooth muscle cell pairs (A7r5) and in rat mesenteric small artery segments simultaneously with force. In A7r5 coupling between cells was estimated by measuring membrane capacitance. Smooth muscle cells were uncoupled when the Na(+)/K(+)-pump was inhibited either by a low concentration of ouabain, which also caused a localized increase of [Ca(2+)](i) near the membrane, or by ATP depletion. Reduction of Na(+)/K(+)-pump activity by removal of extracellular potassium ([K(+)](o)) also uncoupled cells, but only after inhibition of K(ATP) channels. Inhibition of the Na(+)/Ca(2+)-exchange activity by SEA0400 or by a reduction of the equilibrium potential (making it more negative) also uncoupled the cells. Depletion of intracellular Na(+) and clamping of [Ca(2+)](i) at low concentrations prevented the uncoupling. The experiments suggest that the Na(+)/K(+)-pump may affect gap junction conductivity via localized changes in [Ca(2+)](i) through modulation of Na(+)/Ca(2+)-exchanger activity.     

LPS-induced impairment of Na+/K+-ATPase activity: ameliorative effect of tannic acid in mice

Metabolic Brain Disease - Acetylcholine is an excitatory neurotransmitter that modulates synaptic plasticity and communication, and it is essential for learning and memory processes. This...     

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid

The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.     

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.     

Homology modeling of the cation binding sites of Na+K+-ATPase

Homology modeling of the alpha-subunit of Na+K+-ATPase, a representative member of P-type ion transporting ATPases, was carried out to identify the cation (three Na+ and two K+) binding sites in the transmembrane region, based on the two atomic models of Ca2+-ATPase (Ca2+-bound form for Na+, unbound form for K+). A search for potential cation binding sites throughout the atomic models involved calculation of the valence expected from the disposition of oxygen atoms in the model, including water molecules. This search identified three positions for Na+ and two for K+ at which high affinity for the respective cation is expected. In the models presented, Na+- and K+-binding sites are formed at different levels with respect to the membrane, by rearrangements of the transmembrane helices. These rearrangements ensure that release of one type of cation coordinates with the binding of the other. Cations of different radii are accommodated by the use of amino acid residues located on different faces of the helices. Our models readily explain many mutational and biochemical results, including different binding stoichiometry and affinities for Na+ and K+.     

Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.