The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase ␣2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase ␣ 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 membranedelimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation. The nicotinic acetylcholine receptor (nAChR)2 and the Na,K-ATPase are integral membrane proteins that play key roles in membrane excitation. We previously identified a regulatory mechanism, termed acetylcholine (ACh)-induced hyperpolarization, whereby the nAChR and the Na,K-ATPase functionally interact to modulate the membrane potential of rat skeletal muscle (1-4). In this interaction, the binding of nanomolar concentrations of ACh to the nAChR stimulates electrogenic transport by the Na,K-ATPase ␣2 isozyme, causing a membrane hyperpolarization of about Ϫ4 mV. This effect requires prolonged exposure to nanomolar concentrations of nicotinic agonist. This property distinguishes it from the more well characterized, rapid action of micromolar concentrations of ACh, which open the nAChR and produce membrane depolarization (5). This finding suggested that a non-conducting conformation of the nAChR, rather than the open state, is involved in signaling to the Na,K-ATPase. In addition, it was shown that the nAChR and Na,K-ATPase can reciprocally interact in a membrane preparation from the Torpedo electric organ (1), a muscle-derived tissue that is rich in muscle nAChRs and Na,K-ATPase. This finding suggested that the nAChR and Na,K-ATPase may interact as part of a membrane-associated regulatory complex.Importantly, this regulation of Na,K-ATPase activity by the nAChR operates under the physiological conditions of normal muscle use. Its ACh concentration dependence is in the range of the residual ACh concentrations that remain in the muscle interstitial spaces for some time following nerve excitation, and to the ACh concentrations that arise at the neuromuscular junction (NMJ) from non-quantal ACh release. The later have also been shown to activate the Na,K-ATPase and hyperpolarize the end plate membrane (6, 7). Notably, this hyperpolariza-* This work was supported, in whole or in part, by National Institutes of Health

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca 2ϩ imaging analysis, we report that ouabain, a specific ligand of the Na ϩ ,K ϩ -ATPase cardiac glycoside binding site, can prevent glutamate receptor agonistinduced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 M N-methyl-Daspartate (NMDA) or kainate caused apoptosis in ϳ50% 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 current's frequency and the intracellular Ca 2ϩ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid or inhibition of the plasma membrane Na ϩ ,Ca 2ϩ -exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabain's effects on NMDA-or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca 2ϩ extrusion from neurons via the Na ϩ ,Ca 2ϩ exchanger. Because intracellular Ca 2ϩ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca 2ϩ extrusion abolishes its development. These antiapoptotic effects are independent of Na ϩ ,K ϩ -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 ϩ ,K ϩ -ATPase in neuroprotection.

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