Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle

Transverse tubule vesicles isolated from frog skeletal muscle display sodium-calcium exchange activity, which was characterized measuring 45Ca influx in vesicles incubated with sodium. The initial rates of exchange varied as a function of the membrane diffusion potentials imposed across the membrane vesicles, increasing with positive intravesicular potentials according to an electrogenic exchange with a stoichiometry greater than 2 sodium ions per calcium ion transported. The exchange activity was a saturable function of extravesicular free calcium, with an apparent K0.5 value of 3 microM and maximal rates of exchange ranging from 3 to 5 nmol/mg protein per 5 s. The exchange rate increased when intravesicular sodium concentration was increased; saturation was approached when vesicles were incubated with concentrations of 160 mM sodium. The isolated transverse tubule vesicles, which are sealed with the cytoplasmic side out, had a luminal content of 112 +/- 39 nmol calcium per mg protein. In the absence of sodium, the exchanger carried out electroneutral calcium-calcium exchange, which was stimulated by increasing potassium concentrations in the intravesicular side. Calcium-calcium exchange showed an extravesicular calcium dependence similar to the calcium dependence of the sodium-calcium exchange, with an apparent K0.5 of 6 microM. Sodium-calcium and calcium-calcium exchange were both inhibited by amiloride. The sodium-calcium exchange system operated both in the forward and in the reverse mode; sodium, as well as calcium, induced calcium efflux from 45Ca-loaded vesicles. This system may play an important role in decreasing the intracellular calcium concentration in skeletal muscle following electrical stimulation.     

Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle

The distribution of glucose transporters and of insulin receptors on the surface membranes of skeletal muscle was studied, using isolated plasma membranes and transverse tubule preparations. (i) Plasma membranes from rabbit skeletal muscle were prepared according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13862-13871), and transverse tubules from rabbit skeletal muscle were prepared according to Rosemblatt et al. (1981, J. Biol. Chem. 256, 8140-8148) as modified by Hidalgo et al. (1983, J. Biol. Chem. 258, 13937-13945). The membranes were identified by the abundance of nitrendipine receptors in the transverse tubules, and their relative absence from the plasma membranes. (ii) Plasma membranes and transverse tubules were also isolated from rat skeletal muscle, according to a novel procedure that isolates both fractions from the same common homogenate. (iii) Glucose transporters were detected by D-glucose protectable binding of the specific inhibitor [3H]cytochalasin B, and insulin receptors were detected by saturable binding of 125I-insulin. The concentration of glucose transporters was about threefold (rabbit) or fivefold (rat) higher in the transverse tubule membrane compared to the plasma membrane, whereas the insulin receptor concentration was about the same in both membranes. These results indicate that the glucose transporters on the surface of the muscle are preferentially segregated to the transverse tubules, and this poses interesting consequences on the functional response of glucose transport to insulin in skeletal muscle.     

Delayed rectification in the transverse tubules: origin of the late after-potential in frog skeletal muscle

Tetanic stimulation of skeletal muscle fibers elicits a train of spikes followed by a long-lasting depolarization called the late after- potential (LAP). We have conducted experiments to determine the origin of the LAP. Isolated single muscle fibers were treated with a high potassium solution (5 mM or 10 mM K) followed by a sudden reduction of potassium concentration to 2.5 mM. This procedure produced a slow repolarization (K repolarization), which reflects a diffusional outflow of potassium from inside the lumen of the transverse tubular system (T system). Tetanic stimulation was then applied to the same fiber and the LAP was recorded. The time courses of K repolarization and LAP decay were compared and found to be roughly the same. This approximate equality held under various conditions that changed the time courses of both events over a wide range. Both K repolarization and the LAP became slower as fiber radius increased. These results suggest that LAP decay and K repolarization represent the same process. Thus, we conclude that the LAP is caused by potassium accumulation in the T system. A consequence of this conclusion is that delayed rectification channels exist in the T system. A rough estimation suggests that the density of delayed rectification channels is less in the T system than in the surface membrane.     

Isolation of plasma membrane vesicles,derived from transverse tubules,by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$, Mg²⁺-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca²⁺-stimulated, Mg²⁺-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ($\text{800 × g}$) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at $\text{100 000 × g}$ for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ ATPase compared with the other fractions, but it had essentially no Ca²⁺-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.     

Linear electrical properties of the transverse tubules and surface membrane of skeletal muscle fibers

With the use of two intracellular microelectrodes and a circuit designed to compensate for the effects of stray capacitances around the electrodes, transfer impedance measurements were made at frequencies from 0.5 to 1000 c/s on frog sartorius muscle fibers bathed in 7.5 mM K Ringer solution. Complete AC cable analyses performed at 46, 100, 215, 464, and 1000 c/s showed that the fibers behaved as ideal one-dimensional cables having purely resistive internal impedances (R_i = 102 ± 11 Ω cm). Two circuits were considered for fiber inside-outside impedance, a four lumped parameter circuit and a parallel resistance and capacitance shunted by the input impedance of a lattice model for the T-system. Least squares fits to fiber input impedance phase angles were better with the latter circuit than with the former. With the use of the lattice model the specific capacitance of both the surface and transverse tubule membranes was found to be 1 µF/cm² and the internal resistivity of the tubules to be about 300 Ω cm.     

Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media.

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na+ + K+)-stimulated, Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell segments in an amber low-speed (800 x g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100,000 x g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na+ + K+)-stimulated ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined bu a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basememt membrane, could not be detected in this fraction.     

Biogenesis of transverse tubules in skeletal muscle in vitro

The transverse (T) tubules of skeletal muscle are membrane tubules that are continuous with the plasma membrane and penetrate the mature muscle fiber radially to carry surface membrane depolarization to the sites of excitation-contraction coupling. We have studied the development of the T-tubule system in cultured amphibian and mammalian muscle cells using a fluorescent lipid probe and antibodies against T-tubules and plasma membranes. Both the lipid probe and the T-tubule antibody recognized an extensive tubular membrane system which subsequently differentiated into the T-system. At all developmental stages, the molecular composition of the T-system was distinct from that of the plasma membrane, suggesting that during myogenesis T-tubules and the plasma membrane form independently from each other and that exchange of membrane proteins between the two continuous compartments is restricted. In rat muscle cultures, T-tubule-specific antigens were first expressed in terminally differentiated myoblasts. Prior to myoblast fusion the antigens appeared as punctate label throughout the cytoplasm. Shortly after fusion the T-tubule-specific antibody labeled a tubular membrane system that extended from the perinuclear region and penetrated most parts of the cells. In contrast, the lipid probe, which labels the T-tubules by virtue of their direct continuity with the plasma membrane, only labeled short tubules extending from the plasma membrane into the periphery of the myotubes at the early stage in development. Thus, the assembly of the T-tubules appears to begin before their connections with the plasma membrane are established.     

Ion pathways in transverse tubules. Quantification of receptors in membranes isolated from frog and rabbit skeletal muscle

The presence of four cation pathways in membrane vesicles isolated from transverse tubules of frog and rabbit skeletal muscle was studied by measuring binding of specific blockers. Transverse tubules purified from frog muscle have a maximal binding capacity for [3H]nitrendipine (a marker for voltage-dependent calcium channels) of 130 pmol/mg of protein; this binding is strongly dependent on temperature and, at 37 degrees C, on the presence of diltiazem. Receptors for [3H]ethylenediamine tetrodotoxin (a marker for voltage-dependent sodium channels) and for 125I-labeled alpha-bungarotoxin (a marker for acetylcholine-mediated channels) showed maximal binding values of about 5 pmol/mg. The number of sodium-pumping sites in the isolated tubule vesicles, inferred from [3H]ouabain binding, was 215 pmol/mg. The high purity of this preparation makes feasible the use of these values as a criterion to judge the degree of purity of isolated preparations, and it allows investigation of transverse tubule contamination in other muscle membrane fractions.     

Caves and labyrinths: caveolae and transverse tubules in skeletal muscle

Summary Caveolins are small integral membrane proteins with a vital role in the formation and function of caveolae. In this review, the role of caveolin-3, a predominantly muscle-specific member of the caveolin family, will be examined. We speculate that insights into the mechanism of caveolae formation may give clues into the formation of another plasma membrane domain, the transverse-tubule system of muscle cells and propose a role for cholesterol-enriched lipid rafts in this process. In addition, we review recent findings regarding caveolin-3 in differentiated muscle cells and, particularly, in dystrophic muscle.Abbreviations DIG detergent-insoluble glycosphingolipid-enriched complex - DPC dystrophin protein complex - eNOS/nNOS endothelial/neuronal nitric oxide synthase - pTT precursor transverse tubule - T-tubule transverse tubule     

Calcium transport in transverse tubules isolated from rabbit skeletal muscle

Isolated transverse tubule vesicles free of sarcoplasmic reticulum transport calcium with high affinity in the presence of ATP. The calcium transport by transverse tubules differs from calcium transport by sarcoplasmic reticulum. It is not increased by oxalate or phosphate, it has a different temperature dependence, it is inhibited by sub-micromolar concentrations of orthovanadate, it is stimulated by calmodulin, and is inhibited by quercetin without causing calcium release. The rates of calcium transport by transverse tubules are two orders of magnitude lower than those of sarcoplasmic reticulum, suggesting that the calcium pump protein of transverse tubules is a minor component of the membrane. Addition of calmodulin to transverse tubule vesicles--treated with high salt in the presence of EGTA to remove endogenous calmodulin--caused a marked stimulation of transport rates at low concentrations of calcium, and decreased from 1.0 to 0.3 microM the calcium concentration at which half-maximal rates of transport were obtained. A role for the transverse tubule calcium pump in maintaining low sarcoplasmic calcium concentrations is proposed.     

Voltage-dependent calcium channels in skeletal muscle transverse tubules. Measurements of calcium efflux in membrane vesicles

Transverse tubule membranes isolated from rabbit skeletal muscle consist mainly of sealed vesicles that are oriented primarily inside out. These membranes contain a high density of binding sites for 1,4-dihydropyridine calcium channel antagonists. The presence of functional voltage-dependent calcium channels in these membranes has been demonstrated by their ability to mediate 45Ca2+ efflux in response to changes in membrane potential. Fluorescence changes of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine, have shown that transverse tubule vesicles may generate and maintain membrane potentials in response to establishing potassium gradients across the membrane in the presence of valinomycin. A two-step procedure has been developed to measure voltage-dependent calcium fluxes. Vesicles loaded with 45Ca2+ are first diluted into a buffer designed to generate a membrane potential mimicking the resting state of the cell and to reduce the extravesicular Ca2+ to sub-micromolar levels. 45Ca2+ efflux is then measured upon subsequent depolarization. Flux responses are modulated with appropriate pharmacological specificity by 1,4-dihydropyridines and are inhibited by other calcium channel antagonists such as lanthanum and verapamil.     

beta-adrenergic receptor and adenylate cyclase in transverse tubules of skeletal muscle.

Experiments were carried out to clarify the sites of action of beta-adrenergic agonists in skeletal muscle microsomes. Microsomes were fractionated into longitudinal reticulum, terminal cisternae, and isolated transverse tubules. Transverse tubules were selectively labeled and tracked with [3H]ouabain. beta-adrenergic receptor was identified by [3H]dihydroalprenolol binding. Assays of beta-adrenergic receptor, adenylate cyclase, and protein kinase-stimulated phosphorylation showed: 1) beta-adrenergic receptor was detected in transverse tubules with a receptor density of 0.61 pmol/mg of protein. No significant binding was detected in longitudinal reticulum or in terminal cisternae. 2) Isoproterenol-stimulated adenylate cyclase was present in microsomes but was similarly confined to the transverse tubular fraction. The activity of F- stimulated cyclase in transverse tubules was 2.3 nmol/mg of protein/min. 3) No phosphorylation of microsomes by cyclic AMP and protein kinase could be detected. We conclude that the action of epinephrine on skeletal muscle is mediated through receptors and adenylate cyclase in the external membrane.     

Plasma membrane removal in rat skeletal muscle fibers reveals caveolin-3 hot-spots at the necks of transverse tubules

Caveolin-3, the muscle-specific isoform of the caveolae-associated protein caveolin, is often thought to be localized exclusively in the surface membrane in mature fibers and associated with transverse (t)-tubular system only transiently during development. Skeletal muscle fibers present a model where the surface membrane (sarcolemma) can be completely separated from the cell by mechanical dissection. Western blotting of matching portions of individual fibers from adult rat muscle in which the sarcolemma was either removed (skinned segment), or left in place (intact segment), revealed that ≥ 70% of caveolin-3 is actually located deeper in the fiber rather than in the sarcolemma itself. Triton solubility of caveolin-3 was no different between sarcolemmal and t-tubule compartments. Confocal immunofluorescence microscopy showed caveolin-3 present throughout the t-system in adult fibers, with ‘hot-spots’ at the necks of the tubules in the sub-sarcolemmal space. A similar representation was seen for the muscle specific voltage-dependent sodium channel Nav1.4 and it was found that at least some Nav1.4 co-immunoprecipitated with caveolin-3 in skinned muscle fibers. The caveolin-3 hot-spots just inside the opening of t-tubules may form regions that localize ion channels and kinases at the key place needed for efficient electrical transmission into the t-tubules as well as for other signaling processes.     

Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules

The purified calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubule membrane consists of three subunits: alpha with Mr 135 000, beta with Mr 50 000, and gamma with Mr 33 000. Purified receptor preparations were incorporated into phosphatidylcholine (PC) vesicles by addition of PC in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and removal of detergent by molecular sieve chromatography. Forty-five percent of the alpha, beta, and gamma polypeptides and the [3H]dihydropyridine/receptor complex were recovered in association with PC vesicles. The rate of dissociation of the purified and reconstituted dihydropyridine/receptor complex was identical with that in T-tubule membranes, and allosteric modulation by verapamil and diltiazem was retained. The reconstituted calcium antagonist receptor, when occupied by the calcium channel activator BAY K 8644, mediated specific 45Ca2+ and 133Ba2+ transport into the reconstituted vesicles. 45Ca2+ influx was blocked by the organic calcium antagonists PN200-110 (K0.5 = 0.2 microM), D600 (K0.5 = 1.0 microM), and verapamil (K0.5 = 1.5 microM) and by inorganic calcium channel antagonists (La3+ greater than Cd2+ greater than Ni2+ greater than Mg2+) as in intact T-tubules. A close quantitative correlation was observed between the presence of the alpha, beta, and gamma subunits of the calcium antagonist receptor and the ability to mediate 45Ca2+ or 133Ba2+ flux into reconstituted vesicles. Comparison of the number of reconstituted calcium antagonist receptors and functional channels supports the conclusion that only a few percent of the purified calcium antagonist receptor polypeptides are capable of mediating calcium transport as previously demonstrated for calcium antagonist receptors in intact T-tubules.     

Voltage-dependent dynamic FRET signals from the transverse tubules in mammalian skeletal muscle fibers

Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC(4)(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC(4)(5). The peak DeltaF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC(4)(5) exhibit DeltaF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.     

ATP-energized Ca2+ pump in isolated transverse tubules of skeletal muscle

A modified protocol for isolation of transverse tubules incorporated an extra stage of purification. The existence of an ATP-energized Ca2+ pump in transverse tubules isolated from rabbit skeletal muscle has been demonstrated. Isolated transverse tubules had a Ca-ATPase activity of 0.78 mu mol/min . mg; this was 300% in excess of that activity attributable to sarcoplasmic reticulum contamination. The distribution of part of the CaATPase activity and ATP-energized Ca2+ uptake coincided with the distribution of transverse tubules in isopycnic sucrose gradients loaded with mechanically disrupted triad junctions. Transverse tubules accumulated over 70 nmol of Ca2+/mg of protein; this uptake was abolished by the Ca2+ ionophore A23187. Neither digitoxin nor monensin inhibited Ca2+ uptake, indicating that Ca2+ accumulation did not occur through a sodium/calcium exchange. Conditions for half-maximal Ca2+ uptake were 5 micro M free Ca2+ and 10 micro M ATP. The Ca2+ pump of isolated transverse tubules was distinguished from the Ca2+ pump of sarcoplasmic reticulum and sarcolemma in that the transverse tubule Ca2+ pump: 1) was not enhanced by oxalate; 2) was not energized by acetyl phosphate, p-nitrophenyl phosphate, or 3-O-methylfluorescein phosphate; and 3) did not hydrolyze p-nitrophenyl phosphate or 3-O-methyl-fluorescein phosphate. Using Ca2+-dependent 3-O-methylfluorescein phosphatase as a marker for sarcoplasmic reticulum, the contamination of the transverse tubule preparation was calculated to be 6%. This agreed with a contamination level of 5% estimated by freeze-fracture electron microscopy.     

Sodium/calcium exchange in amphibian skeletal muscle fibers and isolated transverse tubules

TheNa⁺/Ca²⁺ exchanger participates inCa²⁺ homeostasis in a variety of cells and has a key rolein cardiac muscle physiology. We studied in this work the exchanger ofamphibian skeletal muscle, using both isolated inside-out transversetubule vesicles and single muscle fibers. In vesicles, increasingextravesicular (intracellular) Na⁺ concentrationcooperatively stimulated Ca²⁺ efflux (reverse mode), withthe Hill number equal to 2.8. In contrast to the stimulation of thecardiac exchanger, increasing extravesicular (cytoplasmic)Ca²⁺ concentration ([Ca²⁺]) inhibited thisreverse activity with an IC₅₀ of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at90 mV. Photolysis of a cytoplasmic caged Ca²⁺ compoundactivated an inward current (forward mode) of 23 ± 10 nA(n = 3), with an average current density of 0.6 µA/µF. External Na⁺ withdrawal generated an outwardcurrent (reverse mode) with an average current density of 0.36 ± 0.17 µA/µF (n = 6) but produced a minimal increasein cytosolic [Ca²⁺]. These results suggest that, inskeletal muscle, the main function of the exchanger is to removeCa²⁺ from the cells after stimulation.