Extrajunctional Membrane Research Articles

Short-Term Mild Hypoxia Modulates Na,K-ATPase to Maintain Membrane Electrogenesis in Rat Skeletal Muscle.

The Na,K-ATPase plays an important role in adaptation to hypoxia. Prolonged hypoxia results in loss of skeletal muscle mass, structure, and performance. However, hypoxic preconditioning is known to protect against a variety of functional impairments. In this study, we tested the possibility of mild hypoxia to modulate the Na,K-ATPase and to improve skeletal muscle electrogenesis. The rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Isolated diaphragm and soleus muscles were tested. In the diaphragm muscle, HH increased the α2 Na,K-ATPase isozyme electrogenic activity and stably hyperpolarized the extrajunctional membrane for 24 h. These changes were accompanied by a steady increase in the production of thiobarbituric acid reactive substances as well as a decrease in the serum level of endogenous ouabain, a specific ligand of the Na,K-ATPase. HH also increased the α2 Na,K-ATPase membrane abundance without changing its total protein content; the plasma membrane lipid-ordered phase did not change. In the soleus muscle, HH protected against disuse (hindlimb suspension) induced sarcolemmal depolarization. Considering that the Na,K-ATPase is critical for maintaining skeletal muscle electrogenesis and performance, these findings may have implications for countermeasures in disuse-induced pathology and hypoxic therapy.

Catecholamine-dependent hyperpolarization of the junctional membrane via β2- adrenoreceptor/Gi-protein/α2-Na-K-ATPase pathway

We investigated the effects of catecholamines, adrenaline and noradrenaline, as well as β-adrenoceptor (AR) modulators on a resting membrane potential at the junctional and extrajunctional regions of mouse fast-twitch Levator auris longus muscle. The aim of the study was to find which AR subtypes, signaling molecules and Na,K-ATPase isoforms are involved in the hyperpolarizing action of catecholamines and whether this action could be accompanied by changes in the pump abundance on the sarcolemma. Adrenaline, noradrenaline and specific β2-AR agonist induced hyperpolarization of both junctional and extrajunctional membrane, but the underlying mechanisms were different. In the junctional membrane the hyperpolarization depended on α2 isoform of the Na,K-ATPase and Gi-protein, whereas in the extrajunctional regions the hyperpolarization mainly relied on α1 isoform of Na,K-ATPase and adenylyl cyclase activities. In both junctional and extrajunctional regions, AR activation caused an increase in Na,K-ATPase abundance in the plasmalemma in a protein kinase A-dependent manner. Thus, the compartment-specific mechanisms are responsible for catecholamine-mediated hyperpolarization in the skeletal muscle.

Early Lipid Raft-Related Changes: Interplay between Unilateral Denervation and Hindlimb Suspension.

Muscle disuse and denervation leads to muscle atrophy, but underlying mechanisms can be different. Previously, we have found ceramide (Cer) accumulation and lipid raft disruption after acute hindlimb suspension (HS), a model of muscle disuse. Herein, using biochemical and fluorescent approaches the influence of unilateral denervation itself and in combination with short-term HS on membrane-related parameters of rat soleus muscle was studied. Denervation increased immunoexpression of sphingomyelinase and Cer in plasmalemmal regions, but decreased Cer content in the raft fraction and enhanced lipid raft integrity. Preliminary denervation suppressed (1) HS-induced Cer accumulation in plasmalemmal regions, shown for both nonraft and raft-fractions; (2) HS-mediated decrease in lipid raft integrity. Similar to denervation, inhibition of the sciatic nerve afferents with capsaicin itself increased Cer plasmalemmal immunoexpression, but attenuated the membrane-related effects of HS. Finally, both denervation and capsaicin treatment increased immunoexpression of proapoptotic protein Bax and inhibited HS-driven increase in antiapoptotic protein Bcl-2. Thus, denervation can increase lipid raft formation and attenuate HS-induced alterations probably due to decrease of Cer levels in the raft fraction. The effects of denervation could be at least partially caused by the loss of afferentation. The study points to the importance of motor and afferent inputs in control of Cer distribution and thereby stability of lipid rafts in the junctional and extrajunctional membranes of the muscle.

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain.

While the role of circulating ouabain-like compounds in the cardiovascular and central nervous systems, kidney and other tissues in health and disease is well documented, little is known about its effects in skeletal muscle. In this study, rats were intraperitoneally injected with ouabain (0.1–10 µg/kg for 4 days) alone or with subsequent injections of lipopolysaccharide (1 mg/kg). Some rats were also subjected to disuse for 6 h by hindlimb suspension. In the diaphragm muscle, chronic ouabain (1 µg/kg) hyperpolarized resting potential of extrajunctional membrane due to specific increase in electrogenic transport activity of the α2 Na,K-ATPase isozyme and without changes in α1 and α2 Na,K-ATPase protein content. Ouabain (10–20 nM), acutely applied to isolated intact diaphragm muscle from not injected rats, hyperpolarized the membrane to a similar extent. Chronic ouabain administration prevented lipopolysaccharide-induced (diaphragm muscle) or disuse-induced (soleus muscle) depolarization of the extrajunctional membrane. No stimulation of the α1 Na,K-ATPase activity in human red blood cells, purified lamb kidney and Torpedo membrane preparations by low ouabain concentrations was observed. Our results suggest that skeletal muscle electrogenesis is subjected to regulation by circulating ouabain via the α2 Na,K-ATPase isozyme that could be important for adaptation of this tissue to functional impairment.

Activity-dependent vs. neurotrophic modulation of acetylcholine receptor expression: Evidence from rat soleus and extensor digitorum longus muscles confirms the exclusive role of activity.

Evoked electrical muscle activity suppresses the transcription of mRNAs for acetylcholine receptors in extrajunctional myonuclei. Muscle denervation or disuse releases such inhibition and extrajunctional receptors appear. However, in soleus muscles paralysed with nerve-applied tetrodotoxin, a restricted perijunctional region has been described where myonuclei remain inhibited, a finding attributed to nerve-derived trophic factor(s). Here, we reinvestigate extrajunctional acetylcholine receptor expression in soleus and extensor digitorum longus muscles up to 90days after denervation or up to 20days of disuse, to clarify the role of trophic factors, if any. The perijunctional region of soleus muscles strongly expressed acetylcholine receptors during the first 2-3weeks of denervation. After 2-3months, this expression had disappeared. No perijunctional expression was seen after paralysis by tetrodotoxin or botulinum toxin A. In contrast, the extensor digitorum longus never displayed suppressed perijunctional acetylcholine receptor expression after any treatment, suggesting that it is an intrinsic property of soleus muscles. Soleus denervation only transiently removed the suppression, and its presence in long-term denervated soleus muscles contradicts any contribution from nerve-derived trophic factor(s). In conclusion, our results confirm that evoked electrical activity is the physiological factor controlling the expression of acetylcholine receptors in the entire extrajunctional membrane of skeletal muscles.

The Response of Denervated Muscle to Long-Term Electrical Stimulation.

Adapted from: Lømo T, Westgaard RH, Hennig R, Gundersen K. The response of denervated muscle to long-term electrical stimulation, In: Carraro U, Angelini C, eds. Proceedings of the First Abano Terme Meeting on Rehabilitation, 1985 August 28-30, Abano Terme, Padova, Italy, An International Symposium, Satellite Meeting of the XIII World Congress of Neurology, Hamburg 1985. Cleup Padova 1985. pp 81–90.

Endplate Na + Channels Loss Impairs Neuromuscular Transmission in Myasthenia Gravis

Myasthenia gravis (MG) is caused by acetylcholine receptor (AChR) antibodies inducing complement-medicated destruction of endplate membrane. Endplate Na+ channels enhance the safety factor for neuromuscular transmission by reducing the threshold depolarization needed to trigger an action potential (EAP). I previously demonstrated that MG antibodies do not directly alter the function of Na+ channels and that Na+ channels are lost by complement-mediated loss of endplate membrane. I examined the impact of postsynaptic voltage-gated Na+ channel loss on the SF. Comparing intercostal nerve-muscle preparations from controls and MG patients with moderate (M) or severe (S) MG, I found that endplate AChR loss decreased the endplate potential (EPP) from 40.2 ±1.3 to 30.1±1.3 mV (M) and 23.5 ±1.7 (S) (p < 0.001 for both). Endplate Na+ channel loss increased EAP from −71.9 ±2.2 to −66.1 ±2.7 (M; p < 0.01) and 62.3 ±2.7 mV (S; p < 0.001). In contrast, on extrajunctional membrane (> 0.1 cm from the endplate), EAP was the same for MG fibers and controls. The SF for neuromuscular transmission was reduced from 2.98 to 1.58 (M) and 1.09 (S). If the EPP was the same as control, the SF for MG fibers would have been 2.12 (M) or 1.86 (S). If EAP for MG fibers were the same as control fibers, the SF for MG fibers would have been 2.23 (M) or 1.74(S). In moderate MG, reduced EPP accounted for 47% of the SF reduction and increased EAP 53% of the SF reduction. In severe MG, reduced EPP accounted for 59% of the SF reduction and increased EAP 40% of the SF reduction. Conclusion: loss of endplate Na+ channels in MG contributes to the reduction in SF to a similar extent as does the loss of endplate AChRs.

Phosphorylation of Extrajunctional Cx43 in Ischemic-Preconditioned Rat Hearts

Ischemic preconditioning (IP) has been found to intensify ischemia-induced spreading of connexin 43 (Cx43) into the extrajunctional plasma membrane of the rat heart. Since ischemia is known to induce connexin dephosphorylation and plasmalemmal permeabilization, we considered whether IP might delay dephosphorylation of also extrajunctional connexin and thus impede myocyte permeabilization. Regional myocardial ischemia of both 15 and 45 min duration with and without IP, respectively, was induced in anesthetized rats. Sections of the hearts were immunostained for phosphorylated and dephosphorylated Cx43. The ratios of immunofluorescence in gap junctions and extrajunctional membranes, respectively, versus myocyte interiors were determined as relative fluorescence units (RFU). IP, however, was not found to reduce gap junctional dephosphorylated Cx43 (normal perfusion: 1.23 +/- 0.06 RFU; 15 min ischemia without and with IP: 1.80 +/- 0.09 and 2.10 +/- 0.24 RFU; 45 min ischemia without and with IP: 3.12 +/- 0.55 and 2.57 +/- 0.33 RFU, respectively). Extrajunctionally, only dephosphorylated Cx43 was found. Its occurrence was not prevented by IP, it was rather intensified by IP (normal perfusion: 1.02 +/- 0.01 RFU, 15 min ischemia without and with IP: 1.06 +/- 0.03 and 1.15 +/- 0.05 RFU (P < 0.02), and 45 min ischemia without and with IP: 1.30 +/- 0.07 and 1.27 +/- 0.07 RFU, respectively). Although under ischemia, junctional and extrajunctional Cx43 predominated in the dephosphorylated state, only a small fraction of myocytes were found permeabilized to propidium iodide. In conclusion, IP did not prevent ischemia-induced Cx43 dephosphorylation in gap junctions and extrajunctional plasma membranes. It is thus more likely that changes in non-channel functions or other localizations of Cx43 are involved in IP-induced myocardial protection.

Early detection of denervated muscle fibers in hindlimb muscles after sciatic nerve transection in wild type mice and in the G93A mouse model of amyotrophic lateral sclerosis

The cell adhesion molecule N-CAM is localized to the adult neuromuscular junction but is also expressed in the extrajunctional membrane of denervated muscles concurrent with extrajunctional acetylcholine receptors. Here we used N-CAM immunohistochemistry to determine whether we could detect early denervation in hindlimb muscles of the G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS). In denervated wild type mouse muscles, N-CAM immunoreactivity on the sarcolemma of all fiber types and within the sarcoplasm of only type IIA fibers was detected at day 2: ∼30% of the muscle fibers in cross-section were fully circumscribed by N-CAM immunoreactivity and ∼25% of fibers were incompletely circumscribed. The proportion of the latter fibers remained constant over the next 8 days as the proportions of the former fibers increased exponentially. Thereafter, fully circumscribed muscle fibers increased to a maximum by 30 days with a concomitant fall in the incompletely circumscribed fibers. Hence, early muscle denervation was detected by the incomplete circumscription of fiber membranes by N-CAM immunoreactivity with full circumscription and intracellular localization indicating more long-term denervation. In the G93A transgenic mouse, rapid denervation of fast-twitch muscles was readily detected by a corresponding proportion of muscle fibers in cross-section with positive N-CAM immunoreactivity. The proportions of incompletely and completely circumscribed muscle fibers corresponded well with the rate of decline in intact motor units and reduced muscle contractile forces. Progressively more fully circumscribed muscle fibers became evident with age. We conclude that the N-CAM immunoreactivity on muscle fiber membranes in muscle cross-sections provides a sensitive means of detecting early muscle fiber denervation.

A Selective Role for MRF4 in Innervated Adult Skeletal Muscle: NaV 1.4 Na+ Channel Expression Is Reduced in MRF4-Null Mice

The factors that regulate transcription and spatial expression of the adult skeletal muscle Na+ channel, Na(V) 1.4, are poorly understood. Here we tested the role of the transcription factor MRF4, one of four basic helix-loop-helix (bHLH) factors expressed in skeletal muscle, in regulation of the Na(V) 1.4 Na+ channel. Overexpression of MRF4 in C2C12 muscle cells dramatically elevated Na(V) 1.4 reporter gene expression, indicating that MRF4 is more efficacious than the other bHLH factors expressed at high levels endogenously in these cells. In vivo, MRF4 protein was found both in extrajunctional and subsynaptic muscle nuclei. To test the importance of MRF4 in Na(V) 1.4 gene regulation in vivo, we examined Na+ channel expression in MRF4-null mice using several techniques, including Western blotting, immunocytochemistry, and electrophysiological recording. By all methods, we found that expression of the Na(V) 1.4 Na+ channel was substantially reduced in MRF4-null mice, both in the surface membrane and at neuromuscular junctions. In contrast, expression of the acetylcholine receptor, and in particular its alpha subunit, was unchanged, indicating that MRF4 regulation of Na+ channel expression was selective. Expression of the bHLH factors myf-5, MyoD, and myogenin was increased in MRF4-null mice, but these factors were not able to fully maintain Na(V) 1.4 Na+ channel expression either in the extrajunctional membrane or at the synapse. Thus, MRF4 appears to play a novel and selective role in adult muscle.

Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.

Utrophin is highly homologous and structurally similar to dystrophin, and in gene delivery experiments in mdx mice was able to functionally replace dystrophin. We performed mini-utrophin gene transfer in Golden Retriever dogs with canine muscular dystrophy (CXMD). Unlike the mouse model, the clinicopathological phenotype of CXMD is similar to that of Duchenne muscular dystrophy (DMD). We injected an adenoviral vector expressing a synthetic utrophin into tibialis anterior muscles of newborn dogs affected with CXMD and examined transgene expression by RNA and protein analysis at 10, 30 and 60 days postinjection in cyclosporin-treated and -untreated animals. Immunosuppression by cyclosporin was required to mitigate the immune response to viral and transgene antigens. RT-PCR analysis showed the presence of the exogenous transcript in the muscle of cyclosporin-treated and -untreated animals. The transgenic utrophin was efficiently expressed at the extrajunctional membrane in immunosuppressed dogs and this expression was stable for at least 60 days. We found reduced fibrosis and increased expression of dystrophin-associated proteins (DAPs) in association with muscle areas expressing the utrophin minigene, indicating that mini-utrophin can functionally compensate for lack of dystrophin in injected muscles. For this reason, utrophin transfer to dystrophin-deficient muscle appears as a promising therapeutic approach to DMD.

Sodium channel distribution on uninnervated and innervated embryonic skeletal myotubes.

Acetylcholine receptor (AChR) and sodium (Na(+)) channel distributions within the membrane of mature vertebrate skeletal muscle fibers maximize the probability of successful neuromuscular transmission and subsequent action potential propagation. AChRs have been studied intensively as a model for understanding the development and regulation of ion channel distribution within the postsynaptic membrane. Na(+) channel distributions have received less attention, although there is evidence that the temporal accumulation of Na(+) channels at developing neuromuscular junctions (NMJs) may differ between species. Even less is known about the development of extrajunctional Na(+) channel distributions. To further our understanding of Na(+) channel distributions within junctional and extrajunctional membranes, we used a novel voltage-clamp method and fluorescent probes to map Na(+) channels on embryonic chick muscle fibers as they developed in vitro and in vivo. Na(+) current densities on uninnervated myotubes were approximately one-tenth the density found within extrajunctional regions of mature fibers, and showed several-fold variations that could not be explained by a random scattering of single channels. Regions of high current density were not correlated with cellular landmarks such as AChR clusters or myonuclei. Under coculture conditions, AChRs rapidly concentrated at developing synapses, while Na(+) channels did not show a significant increase over the 7 day coculture period. In vivo investigations supported a significant temporal separation between Na(+) channel and AChR aggregation at the developing NMJ. These data suggest that extrajunctional Na(+) channels cluster together in a neuronally independent manner and concentrate at the developing avian NMJ much later than AChRs.

In vivo acetylcholine receptor expression induced by calcitonin gene-related peptide in rat soleus muscle.

We applied calcitonin gene-related peptide (CGRP) by continuous perfusion of the extrajunctional surface of the adult rat soleus muscle in vivo. We obtained this through a fine polyethylene catheter connected to an Alzet pump implanted in the animal. The perfusion induced a local acetylcholine receptor accumulation in the membrane of the muscle fibres starting with a delay of one to two days, provided a chronic conduction block of soleus innervation was concomitantly present. The effect was prominent, being higher than that following denervation. The lack of acetylcholine receptor accumulation observed in sham perfused animals and the co-administration of CGRP and its competitive antagonist peptide, hCGRP(8-37), eliminates the possibility that the response to CGRP application represents an inflammatory reaction to foreign bodies instead of a specific effect of the peptide. We suggest that CGRP may act on the extrajunctional membrane of muscle fibres to help induce acetylcholine receptor accumulation after appropriate receptors for the peptide are re-expressed due to muscle paralysis. Whilst this is compatible with a role of CGRP in synaptogenesis, a recent study showed that alpha-CGRP(-/-) mutant mice have normal neuromuscular junction development. However, given the redundancy of factors involved in acetylcholine receptor accumulation, further experiments on multiple knock-outs need to be performed before a final conclusion is reached about the physiological significance of CGRP.

Gq protein alpha subunits Galphaq and Galpha11 are localized at postsynaptic extra-junctional membrane of cerebellar Purkinje cells and hippocampal pyramidal cells.

Following cell surface receptor activation, the alpha subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein alpha subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein alpha subunits, Galphaq and Galpha11 were transcribed predominantly in the brain. The highest transcriptional level of Galphaq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Galpha11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to Galphaq and Galpha11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Galphaq/Galpha11 was extensively colocalized with metabotropic glutamate receptor mGluR1alpha in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1alpha and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771-787; Luján et al. 1996, Eur. J. Neurosci., 8, 1488-1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by Galphaq and/or Galpha11, leading to the activation of the intracellular effector, phospholipase Cbeta.

Electrophysiology of postsynaptic activation.

The safety factor for neuromuscular transmission depends upon the amount of ACh released from the nerve terminal, the number of AChRs, and the concentration of Na+ channels at the end plate potential. The postsynaptic end plate membrane of the neuromuscular junctions is specialized in three ways: (1) AChRs, Na+ channels, ChE, NOS, and other membrane-associated proteins are concentrated at the end plate; (2) the end plate cytoskeleton has a different composition of proteins as compared with extrajunctional membrane; and (3) the end plate membrane is mechanically different as compared with extrajunctional membrane. A blockade of neuromuscular transmission occurs when ACh release is inadequate or the end plate response to ACh is too small to trigger an AP. A safety factor for neuromuscular transmission exists because the EPP is larger than the threshold for generating an AP. The high concentration of Na+ channels at the end plate increases the safety factor for neuromuscular transmission by reducing the threshold depolarization required to initiate an AP. In MG, the safety factor is reduced due to loss of AChRs and loss of Na+ channels. The loss of AChRs reduces the EPP and the Na+ channel loss increases the threshold for triggering an AP.

End-plate voltage-gated sodium channels are lost in clinical and experimental myasthenia gravis.

This study examined the loss of voltage-gated Na+ channels as well as acetylcholine receptors (AChRs) from the end-plate region in patients with acquired myasthenia gravis (MG) and in rats with experimental autoimmune passively transferred MG (PTMG). Rats received a monoclonal IgG antibody directed against an extracellular epitope of the nicotinic acetylcholine receptor of muscle (AChR) to produce PTMG. At the end-plate border we examined miniature end-plate potentials (MEPPs), sodium current (INa) amplitude, and action potential (AP) properties; the latter two were also examined on the extrajunctional membrane. In the normal situation, the safety factor for neuromuscular transmission is ensured by the large INa at the end plate, which reduces the AP threshold. Among different fiber types, INa was largest for type IIb fibers and smallest for type I fibers. When end-plate border properties of fibers from 3 MG patients and 15 PTMG rats were compared with controls, INa was reduced, AP thresholds were higher, and rates of AP rise were reduced. Amplitudes of MEPPs and INa at the end plate indicated that loss of AChRs was greater than loss of Na+ channels in patients with MG and rats with PTMG; INa was reduced to about 60% of control values, whereas MEPPs were reduced to less than 30% of control values. On the extrajunctional membrane, INa and AP thresholds and rates of rise were similar for MG patients, PTMG rats, and controls. This evidence for loss of voltage-gated Na+ channels at the motor end plate in both patients with MG and in rats with PTMG reveals a hitherto unrecognized consequence of the end-plate damage initiated by the binding of complement-fixing IgG to end-plate AChRs.

Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses.

We used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold-silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.

Effects of long-term conduction block on membrane properties of reinnervated and normally innervated rat skeletal muscle.

1. Do motoneurons regulate muscle extrajunctional membrane properties through chemical (trophic) factors in addition to evoked activity? We addressed this question by comparing the effects of denervation and nerve conduction block by tetrodotoxin (TTX) on extrajunctional acetylcholine (ACh) sensitivity and action potential resistance to TTX in adult rats. 2. We applied TTX to sciatic or tibial nerves for up to 5 weeks using an improved blocking technique which completely suppresses conduction but avoids nerve damage. 3. Reinnervation by TTX-blocked axons had no effect on the high ACh sensitivity and TTX resistance induced by nerve crush. 4. Long-lasting block of intact nerves (up to 38 days) induced extrajunctional changes as pronounced as after denervation. At shorter times (3 days), however, denervation induced much larger changes than TTX block; such a difference is thus only transiently present in muscle. 5. The effects of long-lasting block were dose dependent. Dose levels (6.6 micrograms day-1) corresponding to those used in the literature to block the rat sciatic nerve induced muscle effects much smaller than those induced by denervation, confirming published data. Our novel finding is that equal effects are obtained using doses substantially higher (up to 10.5 micrograms day-1). For the soleus it was necessary in addition to apply the TTX directly to the smaller tibial nerve. 6. The TTX-blocked nerves were normal in their histological appearance and capacity to transport anterogradely 3H-labelled proteins, to release ACh in quantal and non-quantal form or cluster ACh receptors and induce functional ectopic junctions on denervated soleus muscles. 7. We conclude that muscle evoked activity is the physiological regulator of extrajunctional membrane properties. Chemical factors from the nerve do not appear to participate in this regulation. The stronger response to denervation at short times only is best accounted for by factors produced by degenerating nerves.

Effects of length changes on Na+ current amplitude and excitability near and far from the end-plate.

Na+ current (INa), membrane capacitance (Cm), action potential (AP) properties, and cable properties were studied on the end-plate (E), the end-plate border (EB), and extrajunctional (EJ) membrane of rat fast twitch muscle fibers. INa normalized to Cm, which is proportional to the density of Na+ channels, was the same on the E and the EB and smallest on EJ membrane. The AP threshold was lower and rate of rise of the AP was larger at the EB compared with EJ membrane. On the E and the EB, Cm and INa did not change in response to changes in fiber length. On EJ membrane, INa, Cm, and membrane cable properties changed in a manner consistent with folding and unfolding of the sarcolemma during length changes. The stiffness of the E membrane may add mechanical stability of the neuromuscular junction so that the electrical properties of the end-plate do not change with fiber length. The higher density of Na+ channels near the end-plate increases the safety factor for neuromuscular transmission by lowering the AP threshold.

Single-channel basis of slow inactivation of Na+ channels in rat skeletal muscle

This study examined the single-channel basis of slow inactivation of Na+ currents (INa) in rat fast-twitch skeletal muscle fibers. A loose patch voltage clamp monitored changes in the maximum inward INa as the holding potential of the membrane patch changed. On a neighboring region of extrajunctional membrane of the same fiber, a gigaohm seal patch voltage clamp recorded single-channel INa. The maximum number of simultaneously open Na+ channels among a group of current traces indicated the maximum number of excitable channels. The holding potentials of the two voltage clamps were the same. Slow inactivation did not affect the open time or conductance of single Na+ channels. The number of excitable Na+ channels reversibly decreased during development of slow inactivation of INa and increased during recovery from slow inactivation of INa. Different stimulation protocols examined whether Na+ channels had to be in the closed, open, or fast-inactivated states to enter the slow-inactivated state. Na+ channels appear to be able to enter the slow-inactivated state from the closed, open, or fast-inactivated state.