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

Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The molecular signals elicited in response to acute and chronic exercise and the integration of multiple intracellular pathways are incompletely understood. We determined the effect of 10 days of intensified cycle training on signal transduction in nine inactive males in response to a 1-h acute bout of cycling at the same absolute workload (164 +/- 9 W). Muscle biopsies were taken at rest and immediately and 3 h after the acute exercise. The metabolic signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), demonstrated divergent regulation by exercise after training. AMPK phosphorylation increased in response to exercise ( approximately 16-fold; P < 0.05), which was abrogated posttraining (P < 0.01). In contrast, mTOR phosphorylation increased in response to exercise ( approximately 2-fold; P < 0.01), which was augmented posttraining (P < 0.01) in the presence of increased mTOR expression (P < 0.05). Exercise elicited divergent effects on mitogen-activated protein kinase (MAPK) pathways after training, with exercise-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation being abolished (P < 0.01) and p38 MAPK maintained. Finally, calmodulin kinase II (CaMKII) exercise-induced phosphorylation and activity were maintained (P < 0.01), despite increased expression ( approximately 2-fold; P < 0.05). In conclusion, 10 days of intensified endurance training attenuated AMPK, ERK1/2, and mTOR, but not CaMKII and p38 MAPK signaling, highlighting molecular pathways important for rapid functional adaptations and maintenance in response to intensified endurance exercise and training.

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

Benziane B, Widegren U, Pirkmajer S, Henriksson J, Stepto NK, Chibalin AV. Effect of exercise and training on phospholemman phosphorylation in human skeletal muscle.

Background: Contractions activate the sodium pump, Na ϩ ,K ϩ -ATPase, and the energy sensor, AMP-activated protein

Apical bumetanide-sensitive Na؉ -K ؉ -2Cl ؊ co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

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.

Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training. Nine untrained, healthy participants (mean±SD; VO2peak 44.1±17.6 ml/kg/min) performed a 60 min bout of cycling exercise at 164±18 W (72% of pre-training VO2peak). Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions (6×5 min; 90–100% VO2peak) and six prolonged moderate-intensity sessions (45–90 min; 75% VO2peak). Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO2peak). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA (1.6 fold; P<0.01) and COXIV protein expression (1.5 fold; P<0.05) were increased by training but COXIV protein expression was decreased (20%; P<0.01) by acute exercise pre- and post-training. These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.