Transverse Tubular Membrane Research Articles

Rapid Ca2+ flux through the transverse tubular membrane, activated by individual action potentials in mammalian skeletal muscle

Periods of low frequency stimulation are known to increase the net Ca(2+) uptake in skeletal muscle but the mechanism responsible for this Ca(2+) entry is not known. In this study a novel high-resolution fluorescence microscopy approach allowed the detection of an action potential-induced Ca(2+) flux across the tubular (t-) system of rat extensor digitorum longus muscle fibres that appears to be responsible for the net uptake of Ca(2+) in working muscle. Action potentials were triggered in the t-system of mechanically skinned fibres from rat by brief field stimulation and t-system [Ca(2+)] ([Ca(2+)](t-sys)) and cytoplasmic [Ca(2+)] ([Ca(2+)](cyto)) were simultaneously resolved on a confocal microscope. When initial [Ca(2+)](t-sys) was > or = 0.2 mM a Ca(2+) flux from t-system to the cytoplasm was observed following a single action potential. The action potential-induced Ca(2+) flux and associated t-system Ca(2+) permeability decayed exponentially and displayed inactivation characteristics such that further Ca(2+) entry across the t-system could not be observed after 2-3 action potentials at 10 Hz stimulation rate. When [Ca(2+)](t-sys) was closer to 0.1 mM, a transient rise in [Ca(2+)](t-sys) was observed almost concurrently with the increase in [Ca(2+)](cyto) following the action potential. The change in direction of Ca(2+) flux was consistent with changes in the direction of the driving force for Ca(2+). This is the first demonstration of a rapid t-system Ca(2+) flux associated with a single action potential in mammalian skeletal muscle. The properties of this channel are inconsistent with a flux through the L-type Ca(2+) channel suggesting that an as yet unidentified t-system protein is conducting this current. This action potential-activated Ca(2+) flux provides an explanation for the previously described Ca(2+) entry and accumulation observed with prolonged, intermittent muscle activity.

A retrograde signal from RyR1 alters DHP receptor inactivation and limits window Ca 2+ release in muscle fibers of Y522S RyR1 knock-in mice

Malignant hyperthermia (MH) is a life-threatening hypermetabolic condition caused by dysfunctional Ca(2+) homeostasis in skeletal muscle, which primarily originates from genetic alterations in the Ca(2+) release channel (ryanodine receptor, RyR1) of the sarcoplasmic reticulum (SR). Owing to its physical interaction with the dihydropyridine receptor (DHPR), RyR1 is controlled by the electrical potential across the transverse tubular (TT) membrane. The DHPR exhibits both voltage-dependent activation and inactivation. Here we determined the impact of an MH mutation in RyR1 (Y522S) on these processes in adult muscle fibers isolated from heterozygous RyR1(Y522S)-knock-in mice. The voltage dependence of DHPR-triggered Ca(2+) release flux was left-shifted by approximately 8 mV. As a consequence, the voltage window for steady-state Ca(2+) release extended to more negative holding potentials in muscle fibers of the RyR1(Y522S)-mice. A rise in temperature from 20 degrees to 30 degrees C caused a further shift to more negative potentials of this window (by approximately 20 mV). The activation of the DHPR-mediated Ca(2+) current was minimally changed by the mutation. However, surprisingly, the voltage dependence of steady-state inactivation of DHPR-mediated calcium conductance and release were also shifted by approximately 10 mV to more negative potentials, indicating a retrograde action of the RyR1 mutation on DHPR inactivation that limits window Ca(2+) release. This effect serves as a compensatory response to the lowered voltage threshold for Ca(2+) release caused by the Y522S mutation and represents a novel mechanism to counteract excessive Ca(2+) leak and store depletion in MH-susceptible muscle.

Ryanodine Receptor Activation by Cav1.2 Is Involved in Dendritic Cell Major Histocompatibility Complex Class II Surface Expression

Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little is known concerning its physiological role and activation mechanism. Here we show that dendritic cells also express the Ca(v)1.2 subunit of the L-type Ca(2+) channel and that release of intracellular Ca(2+) via RyR1 depends on the presence of extracellular Ca(2+) and is sensitive to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid increase in expression of major histocompatibility complex II molecules on the surface of dendritic cells, an effect that is also observed upon incubation of mouse BM12 dendritic cells with transgenic T cells whose T cell receptor is specific for the I-A(bm12) protein. Based on the present results, we suggest that activation of the RyR1 signaling cascade may be important in the early stages of infection, providing the immune system with a rapid mechanism to initiate an early response, facilitating the presentation of antigens to T cells by dendritic cells before their full maturation.

Caffeine – a valuable tool in excitation–contraction coupling research

Caffeine – a valuable tool in excitation–contraction coupling research

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 DiBAC4(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 DiBAC4(5). The peak ΔF/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 DiBAC4(5) exhibit ΔF/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.

Ca 2+ sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle

Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular-sarcoplasmic reticulum junctions, and the unit signal is the Ca(2+) spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RyR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca(2+) sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RyR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca(2+)-induced Ca(2+) release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RyR3 isoform. Because RyR3 does not contact voltage sensors, their opening was probably activated by Ca(2+), secondarily to Ca(2+) release through junctional RyR1. Physiologically voltage-controlled Ca(2+) sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca(2+), and a slave parajunctional RyR3 cohort.

Ion channels and ion transporters of the transverse tubular system of skeletal muscle

This review focuses on the electrical properties of the transverse (T) tubular membrane of skeletal muscle, with reference to the contribution of the T-tubular system (TTS) to the surface action potential, the radial spread of excitation and its role in excitation-contraction coupling. Particularly, the most important ion channels and ion transporters that enable proper depolarization and repolarization of the T-tubular membrane are described. Since propagation of excitation along the TTS into the depth of the fibers is a delicate balance between excitatory and inhibitory currents, the composition of channels and transporters is specific to the TTS and different from the surface membrane. The TTS normally enables the radial spread of excitation and the signal transfer to the sarcoplasmic reticulum to release calcium that activates the contractile apparatus. However, due to its structure, even slight shifts of ions may alter its volume, Nernstian potentials, ion permeabilities, and consequently T-tubular membrane potential and excitability.

Uncoupling Store-Operated Ca 2+ Entry and Altered Ca 2+ Release from Sarcoplasmic Reticulum through Silencing of Junctophilin Genes

Junctophilin (JP) mediates the close contact between cell surface and intracellular membranes in muscle cells ensuring efficient excitation-contraction coupling. Here we demonstrate that disruption of triad junction structure formed by the transverse tubular (TT) invagination of plasma membrane and terminal cisternae of sarcoplasmic reticulum (SR) by reduction of JP expression leads to defective Ca 2+ homeostasis in muscle cells. Using adenovirus with small hairpin interference RNA (shRNA) against both JP1 and JP2 genes, we could achieve acute suppression of JPs in skeletal muscle fibers. The shRNA-treated muscles exhibit deformed triad junctions and reduced store-operated Ca 2+ entry (SOCE), which is likely due to uncoupled retrograde signaling from SR to TT. Knockdown of JP also causes a reduction in SR Ca 2+ storage and altered caffeine-induced Ca 2+ release, suggesting an orthograde regulation of the TT membrane on the SR Ca 2+ release machinery. Our data demonstrate that JPs play an important role in controlling overall intracellular Ca 2+ homeostasis in muscle cells. We speculate that altered expression of JPs may underlie some of the phenotypic changes associated with certain muscle diseases and aging.

Optical Imaging and Functional Characterization of the Transverse Tubular System of Mammalian Muscle Fibers using the Potentiometric Indicator di-8-ANEPPS

Potentiometric dyes are useful tools for studying membrane potential changes from compartments inaccessible to direct electrical recordings. In the past, we have combined electrophysiological and optical techniques to investigate, by using absorbance and fluorescence potentiometric dyes, the electrical properties of the transverse tubular system in amphibian skeletal muscle fibers. In this paper we expand on recent observations using the fluorescent potentiometric indicator di-8-ANEPPS to investigate structural and functional properties of the transverse tubular system in mammalian skeletal muscle fibers. Two-photon laser scanning confocal fluorescence images of live muscle fibers suggest that the distance between consecutive rows of transverse tubules flanking the Z-lines remains relatively constant in muscle fibers stretched to attain sarcomere lengths of up to 3.5 microm. Furthermore, the combined use of two-microelectrode electrophysiological techniques with microscopic fluorescence spectroscopy and imaging allowed us to compare the spectral properties of di-8-ANEPPS fluorescence in fibers at rest, with those of fluorescence transients recorded in stimulated fibers. We found that although the indicator has excitation and emission peaks at 470 and 588 nm, respectively, fluorescence transients display optimal fractional changes (13%/100 mV) when using filters to select excitation wavelengths in the 530-550 nm band and emissions beyond 590 nm. Under these conditions, results from tetanically stimulated fibers and from voltage-clamp experiments suggest strongly that, although the kinetics of di-8-ANEPPS transients in mammalian fibers are very rapid and approximate those of the surface membrane electrical recordings, they arise from the transverse tubular system membranes.

Central core disease mutations R4892W, I4897T and G4898E in the ryanodine receptor isoform 1 reduce the Ca2+ sensitivity and amplitude of Ca2+-dependent Ca2+ release.

Three CCD (central core disease) mutants, R4892W (Arg4892-->), I4897T and G4898E, in the pore region of the skeletal-muscle Ca2+-release channel RyR1 (ryanodine receptor 1) were characterized using a newly developed assay that monitored Ca2+ release in the presence of Ca2+ uptake in microsomes isolated from HEK-293 cells (human embryonic kidney 293 cells), co-expressing each of the three mutants together with SERCA1a (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1a). Both Ca2+ sensitivity and peak amplitude of Ca2+ release were either absent from or sharply decreased in homotetrameric mutants. Co-expression of wild-type RyR1 with mutant RyR1 (heterotetrameric mutants) restored Ca2+ sensitivity partially, in the ratio 1:2, or fully, in the ratio 1:1. Peak amplitude was restored only partially in the ratio 1:2 or 1:1. Reduced amplitude was not correlated with maximum Ca2+ loading or the amount of expressed RyR1 protein. High-affinity [3H]ryanodine binding and caffeine-induced Ca2+ release were also absent from the three homotetrameric mutants. These results indicate that decreased Ca2+ sensitivity is one of the serious defects in these three excitation-contraction uncoupling CCD mutations. In CCD skeletal muscles, where a mixture of wild-type and mutant RyR1 is expressed, these defects are expected to decrease Ca2+-induced Ca2+ release, as well as orthograde Ca2+ release, in response to transverse tubular membrane depolarization.

The effect of extracellular tonicity on the anatomy of triad complexes in amphibian skeletal muscle.

Ultrastructural features of tubular-sarcoplasmic (T-SR) triad junctions and measures of cell volume following graded increases of extracellular tonicity were compared under physiological conditions recently shown to produce spontaneous release of intracellularly stored Ca2+ in fully polarized amphibian skeletal muscle fibres. The fibres were fixed using solutions of equivalent tonicities prior to processing for electron microscopy. The resulting anatomical sections demonstrated a partially reversible cell shrinkage corresponding to substantial increases in intracellular solute or ionic strength graded with extracellular tonicity. Serial thin sections through triad structures confirmed the presence of geometrically close but anatomically isolated transverse (T-) tubular and sarcoplasmic reticular (SR) membranes contrary to earlier suggestions for the development of luminal continuities between these structures in hypertonic solutions. They also quantitatively demonstrated accompanying decreases in T-SR distances, increased numbers of sections that showed closely apposed T and SR membranes, tubular luminal swelling and reductions in luminal volume of the junctional SR, all correlated with the imposed increases in extracellular osmolarity. Fully polarized fibres correspondingly showed elementary Ca(2+)-release events ('sparks', in 100 mM-sucrose-Ringer solution), sustained Ca2+ elevations and propagated Ca2+ waves (> or = 350-500 mM sucrose) following exposure to physiological Ringer solutions of successively greater tonicities. These were absent in hypotonic, isotonic or less strongly hypertonic (approximately 50 mM sucrose-Ringer) solutions. Yet exposure to hypotonic solutions also disrupted T-SR junctional anatomy. It increased the tubular diameters and T-SR distances and reduced their area of potential contact. The spontaneous release of intracellularly stored Ca2+ thus appears more closely to correlate with the expected changes in intracellular solute strength or a reduction in absolute T-SR distance rather than disruption of an optimal anatomical relationship between T and SR membranes taking place with either increases or decreases in extracellular tonicity.

An Integrative Model of the Cardiac Ventricular Myocyte Incorporating Local Control of Ca 2+ Release

The local control theory of excitation-contraction (EC) coupling in cardiac muscle asserts that L-type Ca 2+ current tightly controls Ca 2+ release from the sarcoplasmic reticulum (SR) via local interaction of closely apposed L-type Ca 2+ channels (LCCs) and ryanodine receptors (RyRs). These local interactions give rise to smoothly graded Ca 2+-induced Ca 2+ release (CICR), which exhibits high gain. In this study we present a biophysically detailed model of the normal canine ventricular myocyte that conforms to local control theory. The model formulation incorporates details of microscopic EC coupling properties in the form of Ca 2+ release units (CaRUs) in which individual sarcolemmal LCCs interact in a stochastic manner with nearby RyRs in localized regions where junctional SR membrane and transverse-tubular membrane are in close proximity. The CaRUs are embedded within and interact with the global systems of the myocyte describing ionic and membrane pump/exchanger currents, SR Ca 2+ uptake, and time-varying cytosolic ion concentrations to form a model of the cardiac action potential (AP). The model can reproduce both the detailed properties of EC coupling, such as variable gain and graded SR Ca 2+ release, and whole-cell phenomena, such as modulation of AP duration by SR Ca 2+ release. Simulations indicate that the local control paradigm predicts stable APs when the L-type Ca 2+ current is adjusted in accord with the balance between voltage- and Ca 2+-dependent inactivation processes as measured experimentally, a scenario where common pool models become unstable. The local control myocyte model provides a means for studying the interrelationship between microscopic and macroscopic behaviors in a manner that would not be possible in experiments.

G(s) and adenylyl cyclase in transverse tubules of heart: implications for cAMP-dependent signaling.

The transverse tubules are highly specialized invaginations of the cardiac sarcolemmal membrane involved in excitation-contraction (EC) coupling. Several proteins directly involved in EC coupling have been shown to reside either in the transverse tubular membrane or in closely associated structures. With the use of immunofluorescence microscopy, we have found that G(S) and adenylyl cyclase, key elements in the beta-adrenergic signal transduction cascade, are essentially homogeneously distributed throughout the transverse tubular network of isolated rat ventricular myocytes. G(S), in particular, was much more abundant within the transverse tubular membrane than in the peripheral sarcolemma. Furthermore, both proteins are also present in the intercalated disk region. The location of these elements of the cAMP-signaling cascade within a few micrometers of every inotropic target suggests that control and action of this second messenger are quite local. Furthermore, a similar distribution is likely for negatively inotropic receptor systems that oppose G(S)-linked receptors at the level of adenylyl cyclase. Thus, in addition to their role in EC coupling, transverse tubules appear to be the primary site for signaling by inotropic agents.

Regulation of depolarization-induced calcium release from skeletal muscle triads by cyclic AMP-dependent protein kinase.

cAMP activated Ca2+ release from the sarcoplasmic reticulum (SR) triggered by transverse tubular membrane depolarization monitored in a triadic vesicle prepared from rabbit skeletal muscle. A kinetic analysis showed that the activation was in the amounts of released Ca2+ and not in the release rates. This activation was found to be depolarization-dependent, that is, the activation effect of cAMP decreased with an increase in the magnitude of depolarization. Because the activation was abolished by a protein kinase inhibitor, protein phosphorylation by the endogenous cAMP-dependent protein kinase (PKA) was thought to be in effect. On the contrary, caffeine-induced Ca2+ release was not activated by cAMP; however, the exogenous PKA catalytic subunit activated Ca2+ release, and this effect was probably through the phosphorylation of the SR Ca2+ release channel as described elsewhere. These results suggest that the protein, except for the SR Ca2+ release channel, is phosphorylated by endogenous PKA and plays a modulatory role in depolarization-induced Ca2+ release, that is, excitation-contraction coupling of the skeletal muscle.

Effect of Collagenase on Surface Expression of Immunoreactive Fibronectin and Laminin in Freshly Isolated Cardiac Myocytes

The extracellular glycoproteins fibronectin (FN) and laminin (LMN) are ubiquitously expressed in myocardial tissue. These glycoproteins are important for cellular attachment and differentiation of the cardiac myocytes. Utilizing specific antibodies for the detection of FN and LMN, respectively, the distribution of these extracellular proteins was examined in enzymatically isolated adult cardiac myocytes. Immunofluorescence staining of rod-shaped cardiac myocytes revealed only remnants of immunoreactive FN on the cellular surface and in the transverse tubular membrane system. LMN expression, however, was preserved in a raster-like pattern in the cardiac myocytes. In order to study the distribution of these glycoproteins at high resolution, scanning electron microscopy using the backscattered electron mode was combined with immunogold staining and silver-enhancement. In addition, to confirm the immunofluorescence microscopic observations it was shown that FN labelling was restricted to ill-defined extracellular material and that LMN was absent from the intercalated discs of the cardiac myocytes. The hypercontracted cells were characterized by numerous surface protrusions devoid of immunoreactive LMN. Thus, these results indicate that FN and LMN are differently affected by collagenase treatment, and that these changes of glycoprotein expression may influence the normal function of the cardiac myocytes as well as the membrane stability during the development of irreversible cellular lesions.

Potentiation of Depolarization-Induced Calcium Release from Skeletal Muscle Triads by Cyclic ADP-Ribose and Inositol 1,4,5-Trisphosphate

Two second messengers, cyclic ADP-ribose (cADPR) and inositol 1,4,5-trisphosphate (IP3), potentiated the Ca2+release from sarcoplasmic reticulum induced by transverse tubular membrane depolarization monitored in a triadic vesicle prepared from skeletal muscle. However, without depolarization they could not trigger the Ca2+release. On the contrary, only cADPR potentiated caffeine-induced Ca2+release. Because Ca2+releases potentiated by cADPR and IP3were inhibited by 1 μM ruthenium red and 100 μM ryanodine, probably these second messengers potentiated the Ca2+release through ryanodine receptor Ca2+channels. These results suggest that in skeletal excitation-contraction coupling, cADPR and IP3play a role as a potentiator or a modifierin vivo,but both modification pathways are different from each other.

Kinetics of depolarization-induced calcium release from skeletal muscle triads in vitro.

Calcium release from the sarcoplasmic reticulum (SR) depending on depolarization of the transverse tubular membrane (TTM) caused by rapid ionic replacement was measured in skeletal muscle triadic vesicles using a stopped-flow apparatus and Fura-2, a membrane-impermeable Ca2+ indicator. Calcium release was triggered by an increase in the magnitude of depolarization. This Ca2+ release was inhibited by ruthenium red, digoxin and dantrolene, and enhanced by caffeine. Thus, Ca2+ release was found to occur through the SR Ca2+ release channel via TTM depolarization and to be able to cause skeletal muscle contraction. Calcium release curves could be divided into two phases. In contrast to other previous studies, in the fast phase the amount of released Ca2+ increased with an increase in the magnitude of depolarization but the Ca2+ release rate did not; on the other hand, in the slow phase the Ca2+ release rate increased but the amount of Ca2+ did not. Furthermore, the Ca2+ release rate was controlled by the luminal Ca2+ concentration of the SR only in the fast phase. These independent dual kinetics of Ca2+ release were explained by the calsequestrin regulation model.

Membrane domain localization of pH-regulating transporters in frog skeletal muscle membrane vesicles.

The distribution of pH-regulating transporters in surface and transverse (T) tubular membrane (TTM) domains of frog skeletal muscle was studied. 2',7'-Bis(carboxyethyl)-5(6)- carboxyfluorescein-loaded giant sarcolemmal vesicles, containing surface membrane, exhibited reversible Na+/H+ exchange. A microsomal vesicle fraction was shown to be enriched in TTM on the basis of high Na(+)-K(+)-ATPase and Mg(2+)-ATPase activity, high ouabain and nitrendipine binding, and low Ca(2+)-ATPase activity. TTM vesicles were well sealed and oriented inside out. Vesicles were loaded with the pH-sensitive dye pyranine. In response to an inwardly directed Na+ gradient, vesicles displayed virtually no alkalinization unless monensin was present. No pH response to an imposed Na+ gradient was seen regardless of the direction of the pH gradient across the vesicles, after phosphorylation of the vesicles with protein kinase C, or when exposed to guanosine 5'-O-(3-thiotriphosphate). In the presence of CO2, addition of Na+ or Cl- had no effect on vesicle pH. These data indicate that the TTM lacks functional pH-regulating transporters [Na+/H+ and (Na+ + HCO3-)/Cl- exchangers], suggesting that pH-regulating transporters are localized only to the surface membrane domain in frog muscle.

Intracellular pH regulation in detubulated frog skeletal muscle fibers.

Intracellular pH regulation was studied in semitendinosus muscle fibers from frog (Rana pipiens). Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes and membrane potential with conventional microelectrodes. Fibers had their connections between the surface and transverse tubular membrane disrupted (detubulation) with the formamide shock technique. Fibers were approximately 80% detubulated as determined by the decrease in membrane capacitance and the loss of contractile capability. The initial rate of pHi recovery from acidification to approximately 6.8 (no CO2) was dependent on external buffering power, reaching a maximum of approximately 0.6 pH/h at 50 mM HEPES, indicating that the rate of pHi recovery in frog muscle is limited by the diffusion of buffer through an external "unstirred layer". In detubulated fibers, pHi recovery from acidification due to both the amiloride-sensitive Na+/H+ and the 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive (Na+ + HCO3-)/Cl- exchangers was nearly identical to recovery in fully tubulated fibers. This is consistent with these two pH recovery transporters being localized to the surface, and not the transverse tubular, membrane domain in frog skeletal muscle fibers.

Two mechanisms of quantized calcium release in skeletal muscle.

Skeletal muscle uses voltage sensors in the transverse tubular membrane that are linked by protein-protein interactions to intracellular ryanodine receptors, which gate the release of calcium from the sarcoplasmic reticulum. Here we show, by using voltage-clamped single fibres and confocal imaging, that stochastic calcium-release events, visualized as Ca2+ sparks, occur in skeletal muscle and originate at the triad. Unitary triadic Ca(2+)-release events are initiated by the voltage sensor in a steeply voltage-dependent manner, or occur spontaneously by a mechanism independent of the voltage sensor. Large-amplitude events also occur during depolarization and consist of two or more unitary events. We propose a 'dual-control' model for discrete Ca2+ release events from the sacroplasmic reticulum that unifies diverse observations about Ca(2+)-signalling in frog skeletal muscle, and that may be applicable to other excitable cells.