Angela F. Dulhunty

Distribution of Potassium and Chloride Permeability over the Surface and T-Tubule Membranes of Mammalian Skeletal Muscle

SummaryThe distribution of K and Cl permeability,PK andPCl, over the surface and T-tubule membranes of red rat sternomastoid fibers has been determined. Membrane potential,Vm, was recorded with 3-m KCl-filled glass microelectrodes. Changes inVm with changes in [K]o or [Cl]o were used to estimatePCl/PK in normal and detubulated preparations. The results show that the T-tubule membrane has a highPCl and is therefore different from the T-tubule membrane of amphibian fibers. Analysis of the time course of depolarization when [K]o was raised (in SO4 solutions) showed thatPK was distributed over the surface and T-tubule membranes. Two observations suggested that T-tubulePCl was higher than the surfacePCl. Firstly, in normal fibers, the depolarization caused by an increase in [K]o was 3.5 times greater in SO4 solutions than in Cl solutions. In marked contrast, the depolarization in glycerol-treated fibers was independent of [Cl]o. Secondly, the rapid change inVm when [Cl]o was changed was reduced by 80% after glycerol treatment. Both observations suggest thatPCl was low in glycerol-treated fibers.PCl/PK was calculated from theVm data using Goldman, Hodgkin and Katz equations for Na and K or for Na, K, and Cl. In normal fibersPCl/PK=4.5 and in glycerol-treated fibersPCl/PK=0.28. Since it is unlikely that glycerol treatment would increasePK, the reduction in the ratio must follow the loss of Cl permeability “channels” in the T-tubule membrane.

Heterogeneity of T-tubule geometry in vertebrate skeletal muscle fibres

SummaryAverage dimensions of transverse tubules were obtained from electron micrographs of thin sections of mammalian and amphibian skeletal muscle fibres and the effect of transverse tubule geometry on the electrical characteristics of the fibres has been considered. The preparations examined were toad sartorius, mouse soleus, rat extensor digitorum longus, soleus and sternomastoid muscles. The T-tubule dimensions varied considerably between the different preparations and the average volume to surface ratio of the transverse tubule in amphibian fibres (8.1 nm) was generally greater than that in mammalian fibres (3.0–6.2 nm). The small volume to surface ratio of the mammalian transverse tubule would tend to reduce the electrical space constant of the transverse tubule system and reduce the rate of cross-sectional activation of the fibres during a twitch contraction. The area of transverse tubule membrane in junctional contact with the sarcoplasmic reticulum was determined and was found to be greater in mammalian fibres than in amphibian fibres. The relative areas of junctional contact, along a unit length of transverse tubule, were the same in rat extensor digitorum longus and soleus fibres.

Potassium contractures and mechanical activation in mammalian skeletal muscles

SummaryPotassium (K-) contractures were recorded from slow-twitch (mouse soleus) and fast-twitch (mouse extensor digitorum longus (EDL) and rat sternomastoid) muscles. The mouse limb muscles responded to a maintained increase in external potassium concentration with a rapid increase in tension (fast contracture) which inactivated and was followed by a slow contracture. Rat sternomatoid muscles responded with fast contractures only. The threshold potassium concentration for contraction was higher in fast-twitch muscles than in soleus muscles, at 22 and at 37°C. After corrections had been made for the more rapid depolarization of soleus fibers, the threshold potential for soleus fiber contraction was 15 mV closer to the resting membrane potential than the threshold for fast-twitch fiber contraction. The K-contracture results were confirmed by two microelectrode voltage-clamp experiments. Activation of fast twitch fibers required depolarizing pulses that were 15 to 20 mV greater than the pulses required to activate soleus fibers. When the time courses of K-contractures were compared it was evident that inactivation with prolonged depolarization was much faster in the fast-twitch muscles than in the soleus muscles. The results suggest that the voltage dependence and kinetics of the process coupling T-tubule depolarization with calcium release from the sarcoplasmic reticulum may depend on fiber type in mammalian skeletal muscle.

The contractile properties, histochemistry, ultrastructure and electrophysiology of the cricothyroid and posterior cricoarytenoid muscles in the rat

SummaryThe contractile, histochemical, morphological and electrophysiological properties of two rat laryngeal muscles, the cricothyroid and posterior cricoarytenoid, have been measured. Both muscles act during respiration to maintain upper airway patency and an even distribution of air in the lungs. The cricothyroid and posterior cricoarytenoid are fast-twitch muscles, having contraction times of 3.4 and 7.2 ms respectively, high myosin ATPase activity, abundant sarcoplasmic reticulum (with average volumes of 9% and 15%, respectively, of the fibre volume) and T-system membrane (with average areas of 0.4 and 0.5 µm2 µ−3 of fibre). The large areas of T-tubule membrane are reflected in the average specific membrane capacities of 6.5 µF cm−2 to 10.5 µF cm−2, which are high considering the small diameter of the fibres (20–30 µm). Of the two muscles, the posterior cricoarytenoid has the faster contraction time and the more abundant sarcoplasmic reticulum content. In addition, the posterior cricoarytenoid is less resistant to fatigue and demonstrates lower succinic dehydrogenase activity. The fatigability of this muscle, coupled with its general lack of functional reserve, suggest that its failure may contribute to upper airway obstruction during respiratory distress.

The membrane capacity of mammalian skeletal muscle fibres

SummaryMembrane capacity was measured as a function of fibre diameter in mammalian skeletal muscle fibres under normal conditions and under conditions designed to reduce the membrane chloride conductance, i.e. in solutions in which choride ions were replaced by sulphate or methylsulphate ions, or in normal Krebs solutions containing 2,4-dichlorophenoxyacetic acid (2.5mm). The experiments were done on rat sternomastoid, extensor digitorum longus and soleus muscle fibres. The average membrane capacity of fibres in each muscle was greater than normal when chloride conductance was reduced and the slope of the relationship between membrane capacity and fibre diameter increased. The results were consistent with the hypothesis that the space constant of the transverse tubule system in mammalian fibres is normally short because the transverse tubule membrane has a high chloride conductance. The experimental results imply that the space constant of the transverse tubule system was less than 40 µm for fibres in normal Krebs solution and greater than 100 µm for fibres with low membrane chloride conductance. The space constant was calculated using measured geometrical parameters of the transverse tubule, and measured membrane conductance, and the values were close to 20 µm for fibres in normal Krebs solution and between 50 and 120 µm for fibres with low chloride conductance.

Glycerol treatment in mammalian skeletal muscle.

Summary(1) The effects of glycerol-treatment on the ultrastructure, tension, and electrical properties of rat sternomastoid muscle fibers are described. (2) The effect upon the ultrastructure of fibers differed from that previously reported for amphibian fibers, in that the sarcoplasmic reticulum, as well as the T-system, was disrupted. (3) Tension (tetanus and K-contracture) was abolished when preparations were returned to normal Krebs after exposure to a glycerol-Krebs solution (exposure periods were 1 hr in 200–350mm-glycerol or 10–60 min in 350mm-glycerol), although fibers had normal resting membrane potentials and action potentials. (4) Fibers treated for 1 hr with 350mm-glycerol were detubulated when returned to normal Krebs. Specific membrane capacity was reduced and exogenous horseradish peroxidase (HRP) did not penetrate the T-system. (5) Fibers were not detubulated after treatment for 1 hr with 200 to 300mm-glycerol or after treatment for 10 to 30 min with 350mm glycerol. Specific membrane capacity and resistance were normal and HRP entered the T-system. (6) Ultrastructural disruption of the triad junction became progressively more extensive with increasing glycerol concentration used and may be responsible for uncoupling.

Effect of chloride withdrawal on the geometry of the T-tubules in amphibian and mammalian muscle

SummaryThe relative chloride permeabilities of the T-tubule membranes in mammalian (rat sternomastoid) and amphibian (toad sartorius) skeletal muscle fibers have been assessed from the change in volume of the T-tubules during chloride withdrawal from fibers exposed to low extracellular chloride concentrations. A 3.5- to 4.2-fold increase in T-tubule volume was found in mammalian fibers, and this was shown to be independent of the composition of the low chloride solution or the nature of the fixative used in preparation for electron microscopy. The increase in T-tubule volume was transient and was inhibited by factors which block chloride conductance, i.e., low pH, 2,4-dichlorophenoxyacetic acid, and nitrate ions. A small increase (1.48-fold) in T-tubule volume was seen in amphibian fibers when chloride ions were replaced by sulphate ions. No increase in volume was observed in amphibian T-tubules when methyl sulphate ions replaced chloride ions. The results support the idea that the chloride permeability of the T-tubule membrane is significantly higher in mammalian fibers than in amphibian fibers.

Indentations in the terminal cisternae of amphibian and mammalian skeletal muscle fibers

Indentations (hillocks and dimples) in the terminal cisternae of mammalian and amphibian skeletal muscle fibers were studied using freeze-fracture and serial thin-section techniques. The structures were seen in all muscles and had a regular separation from each other and from the T-tubule. Indentations were smaller than fenestrations and formed concavities in, but not macromolecular pores through, the terminal cisternae. The average numbers of indentations in rat muscles (measured along the length of the terminal cisternae, within 150 nm of the triadic junction) varied from 0.9 per micrometer in soleus fibers to 9.6 per micrometer in posterior cricoarytenoid fibers. The average numbers in amphibian sartorius fibers varied from 1.6 to 3.6 per micrometer in muscles from different species. The regular alignment of the indentations along the triad, as well as a close correlation between their numbers and the contractile properties of the muscle, suggest that they function in contractile activation and may represent sites of calcium release from the terminal cisternae.

Indentations in the terminal cisternae of denervated rat EDL and soleus muscle fibers

The effects of denervation on the structure of the triad and longitudinal sarcoplasmic reticulum have been investigated in freeze-fracture replicas of extensor digitorum longus (EDL) and soleus fibers that had been denervated for 2 to 70 days. In EDL fibers the density of indentations along 1 micron of terminal cisternae fell during the first 2 weeks after nerve section, from a normal value of 7.3 +/- 0.2 (mean +/- 1 SEM) to an average value of 2.0 +/- 0.5 in fibers denervated for 16 to 17 days. Denervation did not change the numbers of indentations in soleus fibers. There was a significant change in the orientation of the triads and organization of the longitudinal sarcoplasmic reticulum in denervated EDL and soleus fibers. The effect of denervation on the density of indentations was best correlated with the effect on asymmetric charge movement, when indentation density was compared with twitch contraction time, the voltage sensitivity of tension, and charge movement. The close relationship between indentation density and charge movement provides compelling evidence for a functional link between the two during excitation-contraction coupling.

Upper motor neurone modulation of charge movement and mechanical activation in rat skeletal muscle fibres.

Abstract The characteristics of charge movement and contractile activation are different in soleus (slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscle fibres of rats. Following mid-thoracic spinal cord transection, both characteristics change in soleus fibres to resemble those of EDL fibres, showing that upper motor neurones modulate both contractile activation and charge movement.