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Patch-clamp studies of mammalian skeletal muscle Na(+) channels are commonly done at subphysiological temperatures, usually room temperature. However, at subphysiological temperatures, most Na(+) channels are inactivated at the cell resting potential. This study examined the effects of temperature on fast and slow inactivation of Na(+) channels to determine if temperature changed the fraction of Na(+) channels that were excitable at resting potential. The loose patch voltage clamp recorded Na(+) currents (I(Na)) in vitro at 19, 25, 31, and 37 degrees C from the sarcolemma of rat type IIb fast-twitch omohyoid skeletal muscle fibers. Temperature affected the fraction of Na(+) channels that were excitable at the resting potential. At 19 degrees C, only 30% of channels were excitable at the resting potential. In contrast, at 37 degrees C, 93% of Na(+) channels were excitable at the resting potential. Temperature did not alter the resting potential or the voltage dependencies of activation or fast inactivation. I(Na) available at the resting potential increased with temperature because the steady-state voltage dependence of slow inactivation shifted in a depolarizing direction with increasing temperature. The membrane potential at which half of the Na(+) channels were in the slow inactivated state was shifted by +16 mV at 37 degrees C compared with 19 degrees C. Consequently, the low availability of excitable Na(+) channels at subphysiological temperatures resulted from channels being in the slow, inactivated state at the resting potential.

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.

The effects of iodoacetic acid (IAA) and ischemic contraction were studied in rat extensor digitorum longus muscles. Ischemic stimulation of IAA-treated muscles produced contracture. We measured total muscle water content, distribution of water between intracellular and extracellular spaces, creatine concentration ([Cr]), creatine phosphate concentration ([PCr]), [ATP], [Pi], intracellular pH, and intracellular Ca2+ concentration ([Ca2+]i) at the onset of contracture. [ADP] was calculated from the equilibrium of the creatine kinase reaction using the measured values of [ATP], [PCr], [Cr], and pH. At the onset of contracture there was a 75% reduction of [PCr], a 12-fold increase in [ADP], and an 11-fold increase in [Ca2+]i compared with unstimulated IAA-treated muscles. [ATP] was not depleted at contracture compared with unstimulated IAA-treated muscles, and [Pi] increased less in muscles at contracture compared with stimulated control muscles. The persistent tension in contractures probably resulted from increased [Ca2+]i combined with increased myofibrillar Ca2+ sensitivity due to elevated [ADP] and relatively reduced intracellular acidification and [Pi].

The voltage dependence and amplitude of Na+ currents (INa) were studied with the loose-patch voltage-clamp technique on 19 fast-twitch human intercostal skeletal muscle fibers at the endplate border and > 200 microns from the endplate (extrajunctional). The fibers were histochemically classified as fast-twitch oxidative-glycolytic (type IIa, n = 9) or fast-twitch glycolytic (type IIb, n = 10). The voltage dependence of activation and fast and slow inactivation of INa were similar for membrane patches recorded on the endplate border and on extrajunctional membrane for both fiber types. INa was about fivefold larger on the endplate border compared with extrajunctional membrane for both fiber types. Type IIb fibers had larger values of INa and manifest fast inactivation of INa at more negative potentials than type IIa fibers. The difference between type IIa and IIb fibers may enable IIb fibers to operate at higher firing frequencies for brief periods.

Na current density and membrane capacitance were studied with the loose patch voltage clamp technique on rat fast- and slow-twitch skeletal muscle fibers at three different regions on the fibers: 1) the end plate border, 2) greater than 200 microns from the end plate (extrajunctional), and 3) on the end plate postsynaptic membrane. Fibers were treated with collagenase to improve visualization of the end plate and to enzymatically remove the nerve terminal. The capacitance of membrane patches was similar on fast- and slow-twitch fibers and patches of membrane on the end plate had twice the capacitance of patches elsewhere. For fast- and slow-twitch fibers, the sizes of the Na current normalized to the area of the patch were as follows: end plate greater than end plate border greater than extrajunctional. For both types of fibers, the amplitudes of the Na current normalized to the capacitance of the membrane patch were as follows: end plate approximately end plate border greater than extrajunctional. At each of the three regions, the Na current densities were larger on fast-twitch fibers and fast-twitch fibers had a larger increase in Na current density at the end plate border compared with extrajunctional membrane.

The effects of iodoacetic acid (IAA) and ischemic contraction were studied in rat extensor digitorum longus muscles. Ischemic contraction of IAA-treated muscles produced contracture. The onset of contracture was not associated with a change in sarcolemmal electrical properties or reduction in intracellular [ATP]; however, [creatine phosphate] was reduced by 75% and free [ADP] was increased by 665%. Continued stimulation of IAA-treated fibers resulted in depolarization, loss of membrane excitability, further depletion of creatine phosphate, and reduction in [ATP]. The effects seen in IAA-treated muscle did not appear to result from a direct action of IAA on the surface membrane, contractile proteins, or excitation-contraction coupling. The contractures in IAA-treated muscle may have resulted from increased Ca sensitivity of the contractile proteins, increased myoplasmic [Ca], or both. Both effects may have resulted from increased [ADP]. In addition, the reduced acidification during ischemic contraction of IAA-treated fibers compared with control fibers may have further increased the Ca sensitivity of IAA-treated fibers compared with controls.

Myofibrillar Ca sensitivity and single-channel acetylcholine receptor (AChR) currents were studied in garter snake (Thamnophis sirtalis sirtalis) costocutaneous muscle fibers. Nomarski optics were used to identify tonic and fast- and slow-twitch fibers. Measurements of tension generation were made using chemically skinned fibers. The maximum tensions of the three types of fibers were similar, and the fast- and slow-twitch fibers had similar Ca sensitivities. Compared with twitch fibers, tonic fibers had lower threshold Ca concentrations for tension generation and a larger range of Ca concentrations between threshold and maximum tension. The AChR channels were studied by enzymatically removing the nerve terminals and performing patch-clamp recordings on the exposed postsynaptic membrane. Twitch fibers had only one class of end-plate channel with a conductance of approximately 51 pS. Tonic fibers had two types of synaptic channels. One AChR channel in the tonic fibers resembled the type seen in twitch fibers. The other channel in tonic fibers had a smaller conductance of approximately 33 pS and resembled extrajunctional AChRs on denervated twitch fibers.

We studied the myofibrillar calcium affinity and mechanical threshold of single muscle fibers from the extensor digitorum longus (EDL) and soleus muscles of male Sprague-Dawley rats treated with daily intramuscular injections of dexamethasone, 1.5 mg/kg, for 14 days. The strength-duration and pCa-tension relationships of EDL fibers were not altered by glucocorticoid treatment. However, in soleus fibers the mechanical threshold was increased and the myofibrillar calcium sensitivity was reduced by dexamethasone treatment. Glucocorticoid treatment did not change the maximum tension per cross-sectional area of single-skinned EDL or soleus fibers. The glucocorticoid-induced atrophy and impaired force-generating capacity of fast-twitch muscle are probably not caused by impaired excitation-contraction coupling.

We explored the possibility that glucocorticoid-induced muscle weakness and atrophy resulted from impaired muscle membrane excitability. Male Sprague-Dawley rats received intramuscular injections of dexamethasone, cortisone acetate (equivalent anti-inflammatory doses), or saline for up to 28 days. Temporal patterns of change in muscle mass, twitch and tetanic tension, and membrane potential, cable parameters, and excitability were studied in vitro in the extensor digitorum longus (EDL), soleus (SOL), omohyoid (OMO), caudofemoralis (CF), and the sternomastoid muscles (membrane potential only). the membrane properties of EDL fibers were also studied in vivo (pentobarbital anesthesia). The relative severity of atrophy was OMO greater than CF greater than EDL greater than SOL. Reduction in twitch or tetanic tension never preceded atrophy. The twitch and tetanic tension (per g muscle) increased with glucocorticoid treatment. There were no significant changes in the time course of the twitch or tetanus. Dexamethasone produced more severe atrophy and force reduction than did cortisone acetate. Glucocorticoid treatment produced a depolarization of EDL muscle fibers measured in vitro at 23 degrees C, but this did not appear to be physiologically significant because EDL fibers studied in vivo were not depolarized and had normal action potential amplitudes and thresholds. Glucocorticoid treatment did not change the membrane resistance or capacitance. We conclude that glucocorticoid treatment did not produce muscle weakness by impairing sarcolemmal excitability or excitation-contraction coupling, but that the weakness resulted from muscle atrophy.

This study investigated the influence of muscle source and fiber type on the calcium sensitivity of skinned rat skeletal muscle fibers from predominantly slow muscles [soleus (SOL) and adductor longus (AL)], mixed muscle [posterior gracilis (PG)], and predominantly fast-twitch muscle [extensor digitorum longus (EDL)]. Fibers were characterized histochemically and by one-dimensional protein gel electrophoresis, and calcium-tension relationships were determined. Fiber type and muscle source had significant effects on the negative log of the calcium concentration associated with half-maximal tension (pCa1/2). Slow-twitch fibers had larger values of pCa1/2 than did fast-twitch fibers. Slow-twitch fibers from the predominantly slow muscles, SOL and AL, had similar values of pCa1/2 but slightly smaller values than from the mixed muscle, PG. Fast-glycolytic (FG) fibers from the predominantly fast muscle, EDL, had a higher pCa1/2 than fibers from the mixed fiber type muscle, PG. There were no differences between the pCa1/2 associated with FG and fast-oxidative-glycolytic fibers.