Depolarization-induced Calcium Release Research Articles

CaV1.1 voltage-sensing domain III exclusively controls skeletal muscle excitation-contraction coupling

Skeletal muscle contractions are initiated by action potentials, which are sensed by the voltage-gated calcium channel (CaV1.1) and are conformationally coupled to calcium release from intracellular stores. Notably, CaV1.1 contains four separate voltage-sensing domains (VSDs), which activate channel gating and excitation-contraction (EC-) coupling at different voltages and with distinct kinetics. Here we show that a single VSD of CaV1.1 controls skeletal muscle EC-coupling. Whereas mutations in VSDs I, II and IV affect the current properties but not EC-coupling, only mutations in VSD III alter the voltage-dependence of depolarization-induced calcium release. Molecular dynamics simulations reveal comprehensive, non-canonical state transitions of VSD III in response to membrane depolarization. Identifying the voltage sensor that activates EC-coupling and detecting its unique conformational changes opens the door to unraveling the downstream events linking VSD III motion to the opening of the calcium release channel, and thus resolving the signal transduction mechanism of skeletal muscle EC-coupling.

Role of the JP45-calsequestrin complex on calcium entry in slow twitch skeletal muscles.

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La3+- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca2+ concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca2+ concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.

Role of the JP45-Calsequestrin Complex on Calcium Entry in Slow Twitch Skeletal Muscles

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La3+- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca2+ concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca2+ concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.

Role of Triadin in the Organization of Reticulum Membrane at the Muscle Triad

The terminal cisternae represent one of the functional domains of the skeletal muscle sarcoplasmic reticulum (SR). They are closely apposed to plasma membrane invaginations, the T-tubules, with which they form structures called triads. In triads, the physical interaction between the T-tubule-anchored voltage-sensing channel DHPR and the SR calcium channel RyR1 is essential because it allows the depolarization-induced calcium release that triggers muscle contraction. This interaction between DHPR and RyR1 is based on the peculiar membrane structures of both T-tubules and SR terminal cisternae. However, little is known about the molecular mechanisms governing the formation of SR terminal cisternae. We have previously shown that ablation of triadins, a family of SR transmembrane proteins that interact with RyR1, induced skeletal muscle weakness in knockout mice as well as a modification of the shape of triads. Here we explore the intrinsic molecular properties of the longest triadin isoform Trisk 95. We show that when ectopically expressed, Trisk 95 can modulate reticulum membrane morphology. The membrane deformations induced by Trisk 95 are accompanied by modifications of the microtubule network organization. We show that multimerization of Trisk 95 by disulfide bridges, together with interaction with microtubules, are responsible for the ability of Trisk 95 to structure reticulum membrane. When domains responsible for these molecular properties are deleted, anchoring of Trisk 95 to the triads in muscle cells is strongly decreased, suggesting that oligomers of Trisk 95 and microtubules contribute to the organization of the SR terminal cisternae in a triad.

Amino Acid Residues Gln4020 and Lys4021 of the Ryanodine Receptor Type 1 Are Required for Activation by 4-Chloro-m-cresol

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

Triadin (Trisk 95) Overexpression Blocks Excitation-Contraction Coupling in Rat Skeletal Myotubes

To identify the function of triadin in skeletal muscle, adenovirus-mediated overexpression of Trisk 95 or Trisk 51, the two major skeletal muscle isoforms, was induced in rat skeletal muscle primary cultures, and the physiological behavior of the modified cells was analyzed. Overexpression did not modify the expression level of their protein partners ryanodine receptor, dihydropyridine receptor, and the other triadin. Caffeine-induced calcium release was also unaffected by triadin overexpression. Nevertheless, in the absence of extracellular calcium, depolarization-induced calcium release was almost abolished in Trisk 95 overexpressing myotubes (T95 myotubes), and not modified in Trisk 51 overexpressing myotubes (T51 myotubes). This was not because of a modification of dihydropyridine receptors, as depolarization in presence of external calcium still induced a calcium release, and the activation curve of dihydropyridine receptor was unchanged, in both T95 and T51 myotubes. The calcium release complex was also maintained in T95 myotubes as Trisk 95, ryanodine receptor, dihydropyridine receptor, and Trisk 51 were still co-localized. The effect of Trisk 95 overexpression on depolarization-induced calcium release was reversed by a simultaneous infection with an antisense Trisk 95 adenovirus, indicating the specificity of this effect. Thus, the level of Trisk 95 and not Trisk 51 is important on regulating the calcium release complex, and an excess of this protein can lead to an inhibition of the physiological function of the complex.

The skeletal muscle ryanodine receptor isoform 1 is found at the intercalated discs in human and mouse hearts.

The ryanodine receptors (RyRs) are a class of intracellular calcium release channels of which there are three isoforms. In striated muscle, isoform 1 and isoform 2 are mainly expressed in the terminal cisternae of sarcoplasmic reticulum of skeletal muscle and heart, respectively. Isoform 3 is widely distributed in tissues but in minuscule amounts. These channels release calcium ions from intracellular stores in excitation-contraction coupling for cell signaling. Here, we report the presence of skeletal muscle isoform 1 localized in the intercalated discs (IDs) of human and mouse hearts. By using RyR1 and connexin43 specific antibodies and dual immunofluorescent techniques, both were localized in the proximity of the IDs of human and mouse hearts. We confirmed that RyR1 is localized to the IDs by selective immunoprecipitation of RyR isoform 1 from a subcellular fraction containing IDs from human heart tissue. The functional significance of our observation remains to be elucidated as isoform 1 is involved in depolarization induced calcium release, unlike RyR isoforms 2 and 3 which appear to be involved in calcium induced calcium release.

Direct Visualization of Physiological Calcium-Induced Calcium Release (CICR) Triggered by Calcium Entry via Voltage-Gated Calcium Channels

Physiological Ca-induced Ca release (CICR) is triggered by Ca ions entering the cytoplasm via voltage/ ligand-operated plasmalemmal Ca channels and interacting with Ca-gated Ca channels (ryanodine receptors, RyR) present in the endoplasmic reticulum (ER) membrane. CICR is believed to be significantly involved in shaping neuronal [Ca] i transients [1-3] and is important for neuronal functions including synaptic transmission and plasticity [4]. Here we visualized directly the process of physiological CICR by monitoring simultaneously Ca currents and the concentration of free Ca in the cytosol ([Ca] i ) and within the ER lumen ([Ca] L ). Experiments were performed on cultured neonatal rat dorsal root ganglion neurons by combining a patchclamp whole-cell technique and video imaging. A lowaffinity Ca-sensitive fluorescent dye, Mag-fura-2-AM (K D ~ 50 μM), was used for detecting high-calcium levels inside the ER lumen. The cells were incubated with the dye so it was trapped both inside the intracellular organelles and the cytosol. In order to wash out the cytosolic portion of Mag-fura-2, the whole-cell patchclamp configuration was established. For simultaneous visualization of [Ca] i , the intrapipette solution was supplemented with 0.05 mM high-affinity Ca indicator Fluo-3K 5 . All recordings started when both dyes achieved their steady-state fluorescent level. Mag-fura-2 and Fluo-3 signals were separated using their distinct excitation properties (Ex.340/380 nm for Mag-fura-2 and 488 nm for Fluo-3). The resting [Ca] L varied within the range of 60 to 270 μM (average level 177 ± 58 μM, n = 63). Activation of RyR by brief (5 sec) application of 20 mM caffeine triggered a rapid drop in the ER Ca, which was mirrored by an elevation of Ca in the cytosol. The amplitude of caffeine-induced [Ca] L response varied from 20 to 120 μM; however, we never observed complete depletion of the store in response to short caffeine application. At higher resting [Ca] L , a larger amplitude of Ca decrease was observed. On average, caffeine diminished the resting [Ca] L value by 39 ± 11% (n = 41). Activation of voltage-gated calcium channels by depolarizing the neuron from –70 mV to 0 mV for 3 sec resulted in opposite changes in the fluorescent signals derived from the cytosol and the ER; the [Ca] i elevation was associated with a simultaneous decrease in the [Ca] L . The amplitude of this decrease was smaller than that evoked by caffeine. It varied between 5 and 30 μM and correlated linearly with the resting intraluminal calcium concentration: at higher resting [Ca] L there was a larger Ca drop in response to depolarization. The depolarization-induced calcium release from the ER lumen was potentiated by 1 mM caffeine and completely blocked by 50 μM ryanodine. When Ca entry was graded by varying either the amplitude or the length of the depolarizing pulses, we found that the degree of CICR activation correlated linearly with the amount of Ca ions entering the cytosol. Stimulation of the neuron by a standard I-V protocol resulted in characteristic bell-shaped patterns of [Ca] i transients, which were mirrored by transient [Ca] L decreases. When the duration of depolarizing pulses was varied between

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.

Numerical Methods to Determine Calcium Release Flux from Calcium Transients in Muscle Cells

Several methods are currently in use to estimate the rate of depolarization-induced calcium release in muscle cells from measured calcium transients. One approach first characterizes calcium removal of the cell. This is done by determining parameters of a reaction scheme from a fit to the decay of elevated calcium after the depolarizing stimulus. In a second step, the release rate during depolarization is estimated based on the fitted model. Using simulated calcium transients with known underlying release rates, we tested the fidelity of this analysis in determining the time course of calcium release under different conditions. The analysis reproduced in a satisfactory way the characteristics of the input release rate, even when the assumption that release had ended before the start of the fitting interval was severely violated. Equally good reconstructions of the release rate time course could be obtained when the model used for the analysis differed in structure from the one used for simulating the data. We tested the application of a new strategy (multiple shooting) for fitting parameters in nonlinear differential equation systems. This procedure rendered the analysis less sensitive to ill-chosen initial guesses of the parameters and to noise. A locally adaptive kernel estimator for calculating numerical derivatives allowed good reconstructions of the original release rate time course from noisy calcium transients when other methods failed.

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.

Regulation of Dihydropyridine and Ryanodine Receptor Gene Expression in Skeletal Muscle: ROLE OF NERVE, PROTEIN KINASE C, AND cAMP PATHWAYS

The dihydropyridine (DHP) and ryanodine (RY) receptors play a critical role in depolarization-induced calcium release in skeletal muscle, yet the factors which govern their expression remain unknown. We investigated the roles of electrical activity and trophic factors in the regulation of the genes encoding the alpha 1, alpha 2, and beta subunits of the DHP receptor as well as the RY receptor in rat skeletal muscle in vivo. Muscle paralysis, induced by denervation, had no effect on the DHP receptor mRNA levels while the RY receptor mRNA was decreased. In contrast, chronic superfusion of tetrodotoxin onto the sciatic nerve resulted in a marked increase in mRNA levels and transcriptional activity of both DHP and RY receptor genes. Since nerve can induce changes in second messenger pathways which modulate muscle gene expression, we attempted to identify factors which regulate DHP and RY receptor expression using cultured myotubes. Elevated cAMP levels specifically inhibited the expression of RY receptor mRNA while 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, increased the transcripts encoding the RY receptor and the alpha 1 subunit of the DHP receptor. Changes in the level of mRNAs were paralleled by altered receptor numbers. Neither cAMP nor protein kinase C altered transcriptional activity of the DHP and RY receptor genes. These results demonstrate that neural factor(s) regulate DHP and RY receptor mRNA levels in vivo via transcriptional mechanisms while protein kinase C and cAMP can modulate DHP and RY receptor transcript levels by a transcription-independent process.

The voltage dependence of depolarization-induced calcium release in isolated skeletal muscle triads.

We demonstrate for the first time in this study that triadic vesicles derived from skeletal muscle display a voltage dependence of depolarization-induced calcium release similar to that found in intact muscle. We confirm previous studies by Dunn (1989) which demonstrated that changes in extravesicular potassium induced membrane potential changes in isolated transverse tubules with the voltage sensitive dye DiSC(3)-5. Depolarization-induced calcium release was studied in isolated triadic vesicles through similar changes in extravesicular [K] while clamping extravesicular Ca++ to submicromolar concentrations. The amplitude of fast phase of calcium release, identified as depolarization-induced calcium release, varied with the percentage of transverse tubules in the preparation (determined through 3H-PN200-110 specific activity) and different levels of depolarization. Threshold activation of calcium release was obtained with a 40.5 mV potential change; maximal calcium release was obtained with a 75 to 81 mV potential change. Boltzmann fits to the normalized depolarization induced calcium release plotted against the membrane potential change yielded a voltage dependence (k = 4.5 mV per e-fold change) very similar to that found in intact muscle (k = 3-4 mV per e-fold change; Baylor, Chandler & Marshall 1978, 1983; Miledi et al., 1981). Substitution of methanesulfonate for propionate as the impermeant ion or addition of valinomycin in the depolarizing solutions had little effect on the voltage dependence of calcium release.

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro.

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95-kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential-induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.

Depolarization-induced calcium release from isolated triads measured with impermeant Fura-2

Depolarization-induced Ca2+ release was studied in a mixture of triads and terminal cisternae isolated from rabbit skeletal muscle. The vesicles were actively loaded with known amounts of Ca2+ in the absence of precipitating anions in a solution containing 100 mM K propionate buffer. Changes in extravesicular Ca2+ were monitored with 10 microM Fura-2 (membrane impermeant form). Ca2+ release was initiated by diluting an aliquot of the loaded vesicles into a TEACl release solution designed to maintain a constant [K+].[Cl-] product. Fast release, defined as the percentage of total Ca2+ loaded which released in less than 10 sec, occurred when extravesicular free Ca2+ was in the submicromolar range and was unaffected by 5 mM caffeine under depolarizing conditions, change in external pH to 6.5, and an increase in external Mg2+ concentration from 0.1 to 0.2 mM. Thus, the Ca2+ release measured in these studies is distinct from Ca(2+)-induced Ca2+ release. The fast release more than doubled when a greater dilution (1:20 versus 1:10) of the loaded vesicles into the release solution, which would produce a larger depolarization, was used. The percentage of loaded Ca2+ which released rapidly in a particular triad preparation was similar to the percentage of vesicles structurally coupled as visualized by electron microscopy.

10] Functional and longitudinal sarcoplasmic reticulum of heart muscle

This chapter focuses on the junctional and longitudinal sarcoplasmic reticulum of heart muscle. In heart, muscle contraction is triggered by calcium, which derives from two sources: (1) Extracellular Ca 2+ enters via the plasmalemma during excitation; which then (2) induces Ca 2+ release from sarcoplasmic reticulum (SR). This type of release is referred to as Ca 2+ -induced Ca 2+ release. In fast twitch skeletal muscle, essentially all of the Ca 2+ , which triggers muscle contraction derives from the sarcoplasmic reticulum subsequent to depolarization at the plasmalemma/transverse tubule. This type of release is referred to as "depolarization-induced calcium release. '' In order to study the calcium release mechanism in skeletal muscle, a fraction of junctional terminal cisternae containing well-defined feet structures has been isolated. The feet structures in situ are involved in junctional association with the terminal cisternae to form the triad junction. The calcium release mechanism was found to be modulated by ryanodine at pharmacologically significant concentrations, and has been localized to the terminal cisternae of sarcoplasmic reticulum.

Calcium release from sarcoplasmic reticulum of normal and dystrophic mice

Contraction of skeletal muscle is triggered by release of calcium from the sarcoplasmic reticulum. In this study, highly purified normal and dystrophic mouse sarcoplasmic reticulum vesicles were compared with respect to calcium release characteristics. Sarcoplasmic reticulum vesicles were actively loaded with calcium in the presence of an ATP-regenerating system. Calcium fluxes were followed by dual wavelength spectrophotometry using the metallochromic indicators antipyrylazo III and arsenazo III, and by isotopic techniques. Calcium release from sarcoplasmic reticulum vesicles was elicited by (a) changing the free calcium concentration of the assay medium (calcium-induced calcium release); (b) addition of a permeant anion to the assay medium, following calcium loading in the presence of a relatively impermeant anion (depolarization-induced calcium release); (c) addition of the lipophilic anion tetraphenylboron (TPB −) to the assay medium and (d) using specific experimental conditions, i.e. high phosphate levels and low magnesium (spontaneous calcium release). Drugs known to influence Ca 2+ release were shown to differentially affect the various types of calcium release. Caffeine (10 mM) was found to enhance calcium-induced calcium release from isolated sarcoplasmic reticulum. Ruthenium red (20 μM) inhibited both calcium-induced calcium release and tetraphenylboron-induced calcium release, and partially inhibited spontaneous calcium release and depolarization-induced calcium release. Local anesthetics inhibited spontaneous calcium release in a time-dependent manner, and inhibited calcium-induced calcium release instantaneously, but did not inhibit depolarization-induced calcium release. Use of pharmacological agents indicates that several types of calcium release operate in vitro. No significant differences were found between normal and dystrophic sarcoplasmic reticulum in calcium release kinetics or drug sensitivities.

Depolarization-induced calcium release from sarcoplasmic reticulum fragments. II. Release of calcium incorporated with ATP.

Ca2& incorporated in vesicles of sarcoplasmic reticulum fragments (SRF) by diffusion could be released rapidly by changing the ionic environment, by dilution from methanesulfonate (MS) to chloride. This ion exchange is considered to make the membrane potential of SRF inside-negative. Much faster release of Ca2t was also observed upon osmotic change from high to low. These responses were very similar to the Ca2& release from SRF after take up using ATP, but the release rate was slow in the case of anion exchnage. The behavior of K&, Na&, sucrose, and inulin incorporated in SRF was followed upon similar treatment. These ions and nolecules were not released upon ion exchange, but were immediately released by osomtic treatment. Therefore, the Ca& release upon anion exchange was not due to the bursting of SRF, but to a direct effect such as a membrane potential change of the SRF. The behavior of anion such as C1- and propionate could not followed by the same method because of the large permability of these anions. It was also shown that Ca& release upon ion exchange was not a direct effect of pH change. Liver microsomes did not show Ca& release upon the same treatment as SRF.

Depolarization-induced calcium release from sarcoplasmic reticulum fragments. I. Release of calcium taken up upon using ATP.

Ca2& taken up by sarcoplasmic reticulum membrane fragments (SRF) upon using ATP could be released rapidly by changing the anion outside the vesicles from methanesulfonate to chloride. It is considered that this anion exchange caused depolarization of the sarcoplasmic reticulum membrane. Similar rapid release of Ca2& taken up by SRF was also caused by a change from high to low osmotic pressure, probably due to bursting of the membrane. On the basis of experiments in which these two types of Ca2& release were discriminated, it was concluded that Ca2& bound inside the membrane was released directly by anion exchange (depolarization). However, Ca2& release was not caused by cation exchange. Sucrose inhibited these two types of Ca2& release. Cia2& taken up in the presence of oxalate could not be released by any treatment used. Liver microsome fraction also has Ca2& uptake activity. However, Ca2& was not released upon anion exchange, but was released upon oxmotic change. These results show that Ca2& release from SRF upon anion exchange is specific to the sarcoplasmic reticulum membrane. In conclusion, SRF membrane retains the ability to respond to the depolarization caused by ion exchange and can release the accumulated Ca2&.