I. I. Krivoi

Na+,K+-ATPase Functionally Interacts with the Plasma Membrane Na+,Ca2+ Exchanger to Prevent Ca2+ Overload and Neuronal Apoptosis in Excitotoxic Stress

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca2+ imaging analysis, we report that ouabain, a specific ligand of the Na+,K+-ATPase cardiac glycoside binding site, can prevent glutamate receptor agonist-induced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 μM N-methyl-d-aspartate (NMDA) or kainate caused apoptosis in ∼50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic currents frequency and the intracellular Ca2+ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or inhibition of the plasma membrane Na+,Ca2+-exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabains effects on NMDA- or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca2+ extrusion from neurons via the Na+,Ca2+ exchanger. Because intracellular Ca2+ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca2+ extrusion abolishes its development. These antiapoptotic effects are independent of Na+,K+-ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na+,K+-ATPase in neuroprotection.

On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase

Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na+,K+-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na+,K+-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na+,K+-ATPase were used. Dose–response curves for ouabain, a specific Na+,K+-ATPase inhibitor, were obtained to ascertain which Na+,K+-ATPase isoform(s) is involved. The ACh dose–response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na+,K+-ATPase was studied in purified lamb kidney Na+,K+-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably α2) isoform of Na+,K+-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na+,K+-ATPase. Possible interaction mechanisms are discussed.

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756–765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639–641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase α2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase α2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase α subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.

Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman.

Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21–31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (−4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser63 and Ser68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.

Distinct α2 Na,K-ATPase membrane pools are differently involved in early skeletal muscle remodeling during disuse.

Location, location, location. The Na-K pump of skeletal muscle is regulated differently at neuromuscular junctions.

Specialized Functional Diversity and Interactions of the Na,K-ATPase.

Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations, and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions, and protein kinase signaling pathways. In addition to its “classical” function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids (CTS) triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function.

Role of Cholesterol in the Maintenance of Endplate Electrogenesis in Rat Diaphragm

Methyl-β-cyclodextrin (0.1 mM) reduced resting potential of muscle fibers and abolished local endplate membrane hyperpolarization in rat diaphragm. This effect was associated with selective reduction of electrogenic activity of α2-isoform of Na,K-ATPase without changes in the level of intracellular acetylcholine. Experiments with cholesterol marker filipin showed that methyl-β-cyclodextrin in this dose induced cholesterol translocation from lipid rafts to liquid phase of the membrane without its release into extracellular space. This modification of lipid rafts by methyl-β-cyclodextrin presumably impaired the mechanism maintaining electrogenesis in endplates mediated by modulation of Na,K-ATPase by non-quantum acetylcholine. Cholesterol can serve as a molecular component of this mechanism.

Isoform-Specific Na,K-ATPase Alterations Precede Disuse-Induced Atrophy of Rat Soleus Muscle

This study examines the isoform-specific effects of short-term hindlimb suspension (HS) on the Na,K-ATPase in rat soleus muscle. Rats were exposed to 24–72 h of HS and we analyzed the consequences on soleus muscle mass and contractile parameters; excitability and the resting membrane potential (RMP) of muscle fibers; the electrogenic activity, protein, and mRNA content of the α1 and α2 Na,K-ATPase; the functional activity and plasma membrane localization of the α2 Na,K-ATPase. Our results indicate that 24–72 h of HS specifically decreases the electrogenic activity of the Na,K-ATPase α2 isozyme and the RMP of soleus muscle fibers. This decrease occurs prior to muscle atrophy or any change in contractile parameters. The α2 mRNA and protein content increased after 24 h of HS and returned to initial levels at 72 h; however, even the increased content was not able to restore α2 enzyme activity in the disused soleus muscle. There was no change in the membrane localization of α2 Na,K-ATPase. The α1 Na,K-ATPase electrogenic activity, protein and mRNA content did not change. Our findings suggest that skeletal muscle use is absolutely required for α2 Na,K-ATPase transport activity and provide the first evidence that Na,K-ATPase alterations precede HS-induced muscle atrophy.

Decrease in the electrogenic Contribution of Na,K-ATPase and the resting membrane potential as a possible mechanism of Ca2+ accumulation in rat soleus muscle in a short-term gravity unloading

The resting membrane potential and electrogenic contribution of α1- and α2-isoforms of Na+/K+-ATPase in the rat soleus muscle at early stages of gravity unloading were analyzed. The role of L-type calcium channels in accumulation of calcium ions in the myoplasm under these conditions was estimated. After 3-day antiorthostatic suspension, the resting membrane potential of the muscle fibers decreased from −71.0 ± 0.5 to −66.8 ± 0.7 mV, the muscle excitability reduced, and a trend of muscle fatigue acceleration appeared. The electrogenic contribution of ouabain-sensitive α2-isoform of Na+/K+-ATPase, determined as the depolarization caused by 1μM ouabain, decreased after suspension from 6.2 ± 0.6 to 0.5 ± 0.8 mV. The contribution of ouabain-resistant α1-isoform of Na+/K+-ATPase, determined as an additional depolarization after addition of 500 μM ouabain, decreased from 4.6 ± 0.6 to 2.6 ± 0.6 mV. The intensity of Fluo-4AM fluorescence in individual muscle fibers increased after suspension more than fourfold, which suggests an elevated calcium concentration in the myoplasm. A local delivery of nifedipine, a blocker of the L-type calcium channels, to the muscle removed this effect. The existence of a selective mechanism suppressing the electrogenic contribution of Na+/K+-ATPase α2-isoform, which is the main cause of the muscle fiber membrane depolarization after 3-day suspension, is postulated. The depolarization can activate part of potential-sensitive L-type Ca2+ channels, causing the accumulation of calcium ions in the muscle fiber myoplasm.

Membrane lipid rafts are disturbed in the response of rat skeletal muscle to short-term disuse

Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events that might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6-12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit or fluorescent sterols. In addition, resting intracellular Ca2+ level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na-K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid raft changes in control muscles but was ineffective in suspended muscles, which showed an initial loss of α2 Na-K-ATPase activity. Lipid rafts were able to recover with cholesterol supplementation, suggesting that disturbance results from cholesterol loss. Repetitive nerve stimulation also restores lipid rafts, specifically in the junctional sarcolemma region. Disuse locally lowered the resting intracellular Ca2+ concentration only near the neuromuscular junction of muscle fibers. Our results provide evidence to suggest that the ordering of lipid rafts strongly depends on motor nerve input and may involve interactions with the α2 Na-K-ATPase. Lipid raft disturbance, accompanied by intracellular Ca2+ dysregulation, is among the earliest remodeling events induced by skeletal muscle disuse.