Single Na+ channel currents observed in cultured rat muscle cells

The voltage- and time-dependent conductance of membrane Na+ channels is responsible for the propagation of action potentials in nerve and muscle cells. In voltage-step-clamp experiments on neurone preparations containing 10(4)-10(7) Na+ channels the membrane conductance shows smooth variations in time, but analysis of fluctuations and other eivdence suggest that the underlying single-channel conductance changes are stochastic, rapid transitions between 'closed' and 'open' states as seen in other channel types. We report here the first observations of currents through individual Na+ channels under physiological conditions using an improved version of the extracellular patch-clamp technique on cultured rat muscle cells. Our observations support earlier inferences about channel gating and show a single-channel conductance of approximately 18 pS.     

Na channels in skeletal muscle concentrated near the neuromuscular junction

Neuronal function depends crucially on the spatial segregation of specific membrane proteins, particularly the segregation associated with sites of synaptic contact. Understanding the factors governing this localization of proteins is a major goal of cellular neurobiology. A conspicuous example of synaptic specialization is the almost exclusive localization of vertebrate skeletal muscle acetylcholine (ACh) receptors to the subsynaptic membrane of the neuromuscular junction (for example, refs 1,2). The localization of other membrane proteins in skeletal muscle has been much less studied, but a knowledge of their distribution is crucial for understanding the factors governing regional specialization. We have explored the distribution in muscle of the voltage-gated Na channel responsible for the action potential using the loose patch-clamp technique, and have measured Na currents in 5-10 micron-diameter membrane patches as a function of distance from the end plate region of snake and rat muscle fibres. Here we report that the Na current density immediately adjacent to the endplate is 5-10-fold higher than at regions away from the endplate. The increased Na current density falls off rapidly with distance, reaching the background level 100-200 micron from the endplate. Although one might expect ACh receptors to be concentrated near the region of ACh release, such a concentration for Na channels, which propagate the impulse throughout the length of the cell, is surprising and suggests that factors similar to those responsible for concentrating ACh receptors at the endplate also operate to concentrate Na channels.     

A lethal mutation in mice eliminates the slow calcium current in skeletal muscle cells

Contraction of a vertebrate skeletal muscle fibre is triggered by electrical depolarization of sarcolemmal infoldings termed transverse-tubules (t-tubules), which in turn causes the release of calcium from an internal store, the sarcoplasmic reticulum (SR). The mechanism that links t-tubular depolarization to SR calcium release remains poorly understood. In principle, this link might be provided by the prominent slow calcium current that has been described in skeletal muscle cells of adult frogs and rats. However, blocking this current does not abolish the depolarization-induced contractile responses of frog muscle, and the function of this slow calcium current is unknown. Here we describe measurements of calcium currents in developing skeletal muscle cells of normal rats and mice, and of mice with muscular dysgenesis, a mutation that causes excitation-contraction (E-C) coupling to fail. We find that a slow calcium current is present in skeletal muscle cells of normal animals but absent from skeletal muscle cells of mutant animals. The effect of the mutation is specific to the slow calcium current of skeletal muscle; a fast calcium current is present in developing skeletal muscle cells of both normal and mutant animals, and slow calcium currents are present in cardiac and sensory neurones of mutant animals. We believe this to be the first report of a mutation affecting calcium currents in a multicellular organism. The effects of the mutation raise important questions about the relationship between the slow calcium current and skeletal muscle E-C coupling.     

Purification and reconstitution of the calcium release channel from skeletal muscle

The calcium release channel from rabbit muscle sarcoplasmic reticulum (SR) has been purified and reconstituted as a functional unit in lipid bilayers. Electron microscopy reveals the four-leaf clover structure previously described for the 'feet' that span the transverse tubule (T)-SR junction. Ca2+ release from the SR induced by T-system depolarization during excitation-contraction coupling in muscle may thus be effected through a direct association of the T-system with SR Ca2+-release channels.     

Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.

Purification of skeletal muscle dihydropyridine binding sites has enabled protein complexes to be isolated from which Ca2+ currents have been reconstituted. Complementary DNAs encoding the five subunits of the dihydropyridine receptor, alpha 1, beta, gamma, alpha 2 and delta, have been cloned and it is now recognized that alpha 2 and delta are derived from a common precursor. The alpha 1 subunit can itself produce Ca2+ currents, as was demonstrated using mouse L cells lacking alpha 2 delta, beta and gamma (our unpublished results). In L cells, stable expression of skeletal muscle alpha 1 alone was sufficient to generate voltage-sensitive, high-threshold L-type Ca2+ channel currents which were dihydropyridine-sensitive and blocked by Cd2+, but the activation kinetics were about 100 times slower than expected for skeletal muscle Ca2+ channel currents. This could have been due to the cell type in which alpha 1 was being expressed or to the lack of a regulatory component particularly one of the subunits that copurifies with alpha 1. We show here that coexpression of skeletal muscle beta with skeletal muscle alpha 1 generates cell lines expressing Ca2+ channel currents with normal activation kinetics as evidence for the participation of the dihydropyridine-receptor beta subunits in the generation of skeletal muscle Ca2+ channel currents.     

Acceleration of activation and inactivation by the beta subunit of the skeletal muscle calcium channel.

The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.     

Activation of the epithelial Na+ channel (ENaC) requires CFTR Cl- channel function

It is increasingly being recognized that cells coordinate the activity of separate ion channels that allow electrolytes into the cell. However, a perplexing problem in channel regulation has arisen in the fatal genetic disease cystic fibrosis, which results from the loss of a specific Cl- channel (the CFTR channel) in epithelial cell membranes. Although this defect clearly inhibits the absorption of Na+ in sweat glands, it is widely accepted that Na+ absorption is abnormally elevated in defective airways in cystic fibrosis. The only frequently cited explanation for this hypertransport is that the activity of an epithelial Na+ channel (ENaC) is inversely related to the activity of the CFTR Cl- channel. However, we report here that, in freshly isolated normal sweat ducts, ENaC activity is dependent on, and increases with, CFTR activity. Surprisingly, we also find that the primary defect in Cl- permeability in cystic fibrosis is accompanied secondarily by a Na+ conductance in this tissue that cannot be activated. Thus, reduced salt absorption in cystic fibrosis is due not only to poor Cl- conductance but also to poor Na+ conductance.     

Primary structure of the receptor for calcium channel blockers from skeletal muscle

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.     

Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel.

Skeletal muscle is unusual in that 70-85% of resting membrane conductance is carried by chloride ions. This conductance is essential for membrane-potential stability, as its block by 9-anthracene-carboxylic acid and other drugs causes myotonia. Fish electric organs are developmentally derived from skeletal muscle, suggesting that mammalian muscle may express a homologue of the Torpedo mamorata electroplax chloride channel. We have now cloned the complementary DNA encoding a rat skeletal muscle chloride channel by homology screening to the Cl- channel from Torpedo. It encodes a 994-amino-acid protein which is about 54% identical to the Torpedo channel and is predominantly expressed in skeletal muscle. Messenger RNA amounts in that tissue increase steeply in the first 3-4 weeks after birth, in parallel with the increase in muscle Cl- conductance. Expression from cRNA in Xenopus oocytes leads to 9-anthracene-carboxylic acid-sensitive currents with time and voltage dependence typical for macroscopic muscle Cl- conductance. This and the functional destruction of this channel in mouse myotonia suggests that we have cloned the major skeletal muscle chloride channel.     

IS4 peptide forms ion channel in rat skeletal muscle cell membrane

A 22-mer peptide, identical to the primary sequence of domain I segment 4 (IS4) of rat brain sodium channel I, has been synthesized. IS4 peptide can incorporate into cultured rat skeletal myotube membranes and form ion channels. With patch clamp cell-attached technique single channel currents through IS4 channels can be recorded. The single channel conductances of IS4 channels are distributed heterogeneously. With different holding potentials, the mean open time, the mean closed time and the mean open probability are different respectively. IS4 channels are selective for Na⁺, Li⁺ and K⁺, but not for Cl⁻.     

Endogenous electric field around muscle fibres depends on the Na+-K+ pump

We describe here experiments which reveal a new physiological specialization in the endplate (synaptic) region of skeletal muscle fibres. Using a vibrating microelectrode which can detect small currents flowing in extracellular fluid, we have found that the membrane in the endplate region behaves as though a steady positive current is generated in this location. Current re-enters the fibre in the extrajunctional region. Further experiments show that this current is dependent on the activity of the sodium pump. The electric field created by this current may be important for long-term interactions between muscle and nerve.     

A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle

Receptor-operated Ca2+ entry has been proposed as a signalling mechanism in many cells. Receptor-operated Ca2+ channels (ROCs) were first postulated in smooth muscle by Bolton, van Breemen and Somlyo and Somlyo, but recordings of directly ligand-gated Ca2+ current are lacking. Here we describe receptor-operated Ca2+ current evoked in arterial smooth muscle cells by ATP, a sympathetic neurotransmitter. ATP activates channels with approximately 3:1 selectivity for Ca2+ over Na+ at near-physiological concentrations and with a unitary conductance of approximately 5 pS in 110 mM Ca2+ or Ba2+. The channels can be opened even at very negative potentials and resist inhibition by cadmium or nifedipine, unlike voltage-gated Ca2+ channels; they are not blocked by Mg2+, unlike NMDA (N-methyl-D-aspartate)-activated channels; they are directly activated by ligand, without involvement of readily diffusible second messengers, unlike cation channels in neutrophils and T lymphocytes. Thus, the ATP-activated channels provide a distinct mechanism for excitatory synaptic current and Ca2+ entry in smooth muscle.     

Combined effects of adrenaline and insulin on active electrogenic Na+-K+ transport in rat soleus muscle.

Both beta 2-adrenoreceptor stimulants (such as adrenaline and salbutamol) and insulin can increase active Na+-K+ transport and hyperpolarise skeletal cells. Thus, adrenaline and insulin, which are otherwise antagonistic regulators of several metabolic processes, have one action in common, namely, stimulation of active ion translocation. This is especially interesting as cyclic AMP stimulates Na+-K+ transport, whereas a lowering of the cytoplasmic concentration of cyclic AMP has been proposed as an early signal in the action of insulin. Here we report the results of experiments in which the active Na+-K+ transport and membrane potential (EM) of rat soleus muscles were studied during the action of supramaximal doses of insulin and beta 2-adrenoreceptor stimulants, alone and in combination. We conclude that the stimulant action of insulin on active electrogenic Na+-K+ transport is unlikely to be evoked by a lowering of the intracellular concentration of cyclic AMP.     

Modulation of ATP-sensitive K+ channels in skeletal muscle by intracellular protons

Since their discovery in cardiac muscle, ATP-sensitive K+(KATP) channels have been identified in pancreatic beta-cells, skeletal muscle, smooth muscle and central neurons. The activity of KATP channels is inhibited by the presence of cytosolic ATP. Their wide distribution indicates that they could have important physiological roles that may vary between tissues. In muscle cells the role of K+ channels is to control membrane excitability and the duration of the action potential. In anoxic cardiac ventricular muscle KATP channels are believed to be responsible for shortening the action potential, and it has been proposed that a fall in ATP concentration during metabolic exhaustion increases the activity of KATP channels in skeletal muscle, which may reduce excitability. But the intracellular concentration of ATP in muscle is buffered by creatine phosphate to 5-10 mM, and changes little, even during sustained activity. This concentration is much higher than the intracellular ATP concentration required to half block the KATP-channel current in either cardiac muscle (0.1 mM) or skeletal muscle (0.14 mM), indicating that the open-state probability of KATP channels is normally very low in intact muscle. So it is likely that some additional means of regulating the activity of KATP channels exists, such as the binding of nucleotides other than ATP. Here I present evidence that a decrease in intracellular pH (pHi) markedly reduces the inhibitory effect of ATP on these channels in excised patches from frog skeletal muscle. Because sustained muscular activity can decrease pHi by almost 1 unit in the range at which KATP channels are most sensitive to pHi, it is likely that the activity of these channels in skeletal muscle is regulated by intracellular protons under physiological conditions.     

'Square arrays' in the sarcolemma of human skeletal muscle fibres.

Fast twitch fibres of rat and rabbit show rectangular patterns of intramembrane particles in freeze-fracture preparations of the sarcolemma. These 'square arrays' are almost totally absent in the slow twitch coleus muscle of rat. I report here differences in the incidence of square arrays in human fetal and adult muscle and in different fibres within a human muscle. Square arrays probably classify fast and slow twitch fibres in freeze-fracture preparations of mixed muscles.