Judith A. Heiny

Isochannels and Blocking Modes of Voltage‐Dependent Sodium Channelsa

Our results support the existence of three different Na-channel subtypes or isochannels. These isochannels can be readily distinguished as the predominant Na-channel types in mammalian brain, skeletal muscle, and cardiac muscle. The sensitivity to mu-conotoxin GIIIA and tetrodotoxin is sufficient to classify these channels. The skeletal muscle channel is very sensitive to both tetrodotoxin and mu-conotoxin, the brain channel is sensitive to tetrodotoxin but insensitive to mu-conotoxin, and the heart and denervated muscle channels are insensitive to both toxins. In addition to block at the external receptor site for guanidinium toxins, several other blocking modes can be generalized for batrachotoxin-activated Na channels. One mode is peculiar to certain hydrophobic molecules so far represented by our studies of benzocaine and procaine. These molecules induce discrete blocking events with dwell times that apparently increase with anesthetic concentration and a blocking frequency that increases with negative voltage. This mode is quite distinct from the fast internal block by charged organic molecules that increases with positive voltage. These results imply that it is not possible to ascribe the diverse effects of local anesthetics to a single site in the interior channel mouth, as previously proposed by Hille. Our observations thus support the conclusions of other workers who used mixtures of two local anesthetics to show that the dose-response behavior does not fit single-site behavior, but requires at least two distinct sites. Two additional blocking modes can be distinguished for the interactions of cations at the internal and external mouths of the channel. Organic molecules can apparently enter the electric field from the internal but not the external side of the channel. This result suggests a wide internal entry way to the field and an external constriction that prevents the entry of molecules with a single methyl group but permits entry of divalent inorganic cations such as Ca2+ and Co2+.

Tissue-specific role of the Na,K-ATPase α2 isozyme in skeletal muscle.

Background: The Na,K-ATPase α2 isozyme is the major Na,K-ATPase of skeletal muscles. Results: Its targeted knockout in mouse skeletal muscle impairs contractility and fatigability without change in resting muscle function. Conclusion: The α2 isozyme is regulated by muscle use and enables working skeletal muscles to maintain contraction and resist fatigue. Significance: The Na,K-ATPase α2 isozyme is vital to muscle and movement. The Na,K-ATPase α2 isozyme is the major Na,K-ATPase of mammalian skeletal muscle. This distribution is unique compared with most other cells, which express mainly the Na,K-ATPase α1 isoform, but its functional significance is not known. We developed a gene-targeted mouse (skα2−/−) in which the α2 gene (Atp1a2) is knocked out in the skeletal muscles, and examined the consequences for exercise performance, membrane potentials, contractility, and muscle fatigue. Targeted knockout was confirmed by genotyping, Western blot, and immunohistochemistry. Skeletal muscle cells of skα2−/− mice completely lack α2 protein and have no α2 in the transverse tubules, where its expression is normally enhanced. The α1 isoform, which is normally enhanced on the outer sarcolemma, is up-regulated 2.5-fold without change in subcellular targeting. skα2−/− mice are apparently normal under basal conditions but show significantly reduced exercise capacity when challenged to run. Their skeletal muscles produce less force, are unable to increase force to match demand, and show significantly increased susceptibility to fatigue. The impairments affect both fast and slow muscle types. The subcellular targeting of α2 to the transverse tubules is important for this role. Increasing Na,K-ATPase α1 content cannot fully compensate for the loss of α2. The increased fatigability of skα2−/− muscles is reproduced in control extensor digitorum longus muscles by selectively inhibiting α2 enzyme activity with ouabain. These results demonstrate that the Na,K-ATPase α2 isoform performs an acute, isoform-specific role in skeletal muscle. Its activity is regulated by muscle use and enables working muscles to maintain contraction and resist fatigue.

Inward rectification in the transverse tubular system of frog skeletal muscle studied with potentiometric dyes.

The non‐penetrating potentiometric dyes NK2367 and WW375 were used to investigate the effect of inward rectification on the weighted‐average tubular membrane potential in single frog muscle fibres voltage clamped using a three‐Vaseline‐gap method. In 100 mM‐K solution, when inward rectification was activated by hyperpolarization the steady‐state amplitude of the transverse tubular system (T‐system) optical signal was reduced, and its rise time was faster than that recorded for an equivalent depolarization. The voltage dependence of the optical attenuation followed that of inward rectification, increasing with increasing hyperpolarization. For a voltage‐clamp step of ‐140 mV the optical attenuation was 0.72 which corresponds to a weighted‐average T‐system potential change of 100 mV. When inward rectification was blocked in a Cs, TEA solution the optical attenuation was also abolished. The voltage dependence of the block of the inward currents in solutions containing low concentrations of Cs was also reflected in the T‐system optical signals. Our results were satisfactorily predicted by a radial cable model of the T‐system, assuming the same specific inward rectifier conductance in surface and tubular membranes. This analysis predicts that the measured optical attenuation corresponds to a decrease in the tubular space constant, lambda T, from 120 micron under passive conditions to about 40 micron when inward rectification is fully, activated. The voltage dependence of inward rectification measured at the surface membrane was reasonably well predicted by assuming that the specific conductance obeyed a Boltzmann type of voltage dependence; the major effect of tubular decrements was to reduce the steepness of the total (surface + T‐system) conductance‐voltage relation.

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.

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+.

The K+ affinity of the Na,K-ATPase α2 isoform matches its activity to the range of extracellular K+ concentrations in the T-tubules at rest and during contraction, maintaining the excitability of active muscle.

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.

Optical evidence for a chloride conductance in the T-system of frog skeletal muscle

T-system action potentials were recorded optically from intact frog skeletal muscle fibers stained with the non-penetrating potentiometric dye NK-2367. The effect of chloride removal on the falling phase of the radially propagating tubular action potential was studied to determine whether a chloride conductance located in the T-system membranes contributes to tubular repolarization during activity. Our results show that, in chloride-free Ringer, repolarization of the tubular action potential is significantly slowed. Moreover, the late phase of tubular repolarization is characterized by a large afterpotential, which is highly temperature-dependent and appears as a secondary peak above 10° C. The optical data were compared with predicted T-system action potentials generated from a radial cable equivalent circuit model of the T-system, in which the effects of a distributed tubular leak conductance were tested. Results of this analysis are consistent with the proposal that some of the outward repolarization current during the T-system action potential is drawn across a chloride conductance located in the T-system membranes.

Central Depletion of Brain-Derived Neurotrophic Factor in Mice Results in High Bone Mass and Metabolic Phenotype

Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal differentiation/survival, the regulation of food intake, and the pathobiology of obesity and type 2 diabetes mellitus. BDNF and its receptor are expressed in osteoblasts and chondrocyte. BDNF in vitro has a positive effect on bone; whether central BDNF affects bone mass in vivo is not known. We therefore examined bone mass and energy use in brain-targeted BDNF conditional knockout mice (Bdnf(2lox/2lox)/93). The deletion of BDNF in the brain led to a metabolic phenotype characterized by hyperphagia, obesity, and increased abdominal white adipose tissue. Central BDNF deletion produces a marked skeletal phenotype characterized by increased femur length, elevated whole bone mineral density, and bone mineral content. The skeletal changes are developmentally regulated and appear concurrently with the metabolic phenotype, suggesting that the metabolic and skeletal actions of BDNF are linked. The increased bone development is evident in both the cortical and trabecular regions. Compared with control, Bdnf(2lox/2lox)/93 mice show greater trabecular bone volume (+50% for distal femur, P < 0.001; +35% for vertebral body, P < 0.001) and midfemoral cortical thickness (+11 to 17%, P < 0.05), measured at 3 and 6 months of age. The skeletal and metabolic phenotypes were gender dependent, with female being more affected than male mice. However, uncoupling protein-1 expression in brown fat, a marker of sympathetic tone, was not different between genotypes. We show that deletion of central BDNF expression in mice results in increased bone mass and white adipose tissue, with no significant changes in sympathetic signaling or peripheral serotonin, associated with hyperphagia, obesity, and leptin resistance.

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.

Metal ion transport quantified by ICP-MS in intact cells.

The use of ICP-MS to measure metal ion content in biological tissues offers a highly sensitive means to study metal-dependent physiological processes. Here we describe the application of ICP-MS to measure membrane transport of Rb and K ions by the Na,K-ATPase in mouse skeletal muscles and human red blood cells. The ICP-MS method provides greater precision and statistical power than possible with conventional tracer flux methods. The method is widely applicable to studies of other metal ion transporters and metal-dependent processes in a range of cell types and conditions.