Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

Ca2+ channel agonist BAY-k 8644 does not elicit Ca2+ release from skeletal muscle sarcoplasmic reticulum

BAY-k 8644, a nifedipine analogue, promotes Ca2+ influx into excitable cells via plasma membrane voltage-sensitive Ca2+ channels. We report here that sarcoplasmic reticulum (SR) Ca2+ release channels are insensitive to BAY-k 8644, as studied in highly purified isolated fractions and in chemically skinned fibers of rabbit skeletal muscle. This result suggests that a subcellular heterogeneity exists among Ca2+ channels, at least with respect to drug-receptor sites. In the course of this study, however we found that BAY-k 8644 reversibly inhibits the SR Ca2+ pump, i.e., it decreases Ca2+ influx into the SR lumen, although at concentrations (IC50 = 3-5 X 10(-5) M) much higher than those effective on voltage-sensitive Ca2+ channels.     

Biochemical characterization of the Ca2+ release channel of skeletal and cardiac sarcoplasmic reticulum

Summary Rapid mixing-vesicle ion flux and planar lipid bilayer-single channel measurements have shown that a high-conductance, ligand-gated Ca²⁺ release channel is present in heavy, junctional-derived membrane fractions of skeletal and cardiac muscle sarcoplasmic reticulum. Using the release channel-specific probe, ryanodine, a 30S protein complex composed of polypeptides of M_r 400 000 has been isolated from cardiac and skeletal muscle. Reconstitution of the complex into planar lipid bilayers has revealed a Ca²⁺ conductance with properties characteristic of the native Ca²⁺ release channel.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by palmitoyl carnitine.

Studies of [3H]ryanodine binding, 45Ca2+ efflux, and single channel recordings in planar bilayers indicated that the fatty acid metabolite palmitoyl carnitine produced a direct stimulation of the Ca2+ release channel (ryanodine receptor) of rabbit and pig skeletal muscle junctional sarcoplasmic reticulum. At a concentration of 50 microM, palmitoyl carnitine (a) stimulated [3H]ryanodine binding 1.6-fold in a competitive manner at all pCa in the range 6 to 3; (b) released approximately 65% (30 nmol) of passively loaded 45Ca2+/mg protein; and (c) increased 7-fold the open probability of Ca2+ release channels incorporated into planar bilayers. Neither carnitine nor palmitic acid could reproduce the effect of palmitoyl carnitine on [3H]ryanodine binding, 45Ca2+ release, or channel open probability. 45Ca2+ release was induced by several long-chain acyl carnitines (C14, C16, C18) and acyl coenzyme A derivatives (C12, C14, C16), but not by the short-chain derivative C8 or by free saturated fatty acids of chain length C8 to C18, at room temperature or 36 degrees C. This newly identified interaction of esterified fatty acids and ryanodine receptors may represent a pathway by which metabolism of skeletal muscle could influence intracellular Ca2+ and may be responsible for the pathophysiology of disorders of beta-oxidation such as carnitine palmitoyl transferase II deficiency.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Regulation of the gating of the sheep cardiac sarcoplasmic reticulum ca2+-release channel by luminal Ca2+

We investigated the effects of changes in luminal [Ca²⁺] on the gating of native andpurified sheep cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channels reconstituted intoplanar phospholipid bilayers. The open probability (P _o )of channels activated solely by cytosolic Ca²⁺ was greater at positive than negative holding potentials. Channels activatedsolely by 10 m cytosolic Ca²⁺ exhibited no change in steady-stateP _o or in the relationship betweenP _o and voltage when the luminal[Ca²⁺] was increased from nanomolar to millimolar concentrations. In the absence of activating concentrationsof cytosolic Ca²⁺, the channel can be activated by the phosphodiesterase inhibitor sulmazole (AR-L 115BS). However, cytosolicCa²⁺-independent activation of the channel by sulmazole requires luminal Ca²⁺. In the presence ofsulmazole, at picomolar luminal [Ca²⁺] the channel remains completely closed. Increasing the luminal [Ca²⁺]to millimolar levels markedly increases the P _o via an increase in theduration of open events. The P _o and duration of the sulmazole-activated, luminalCa²⁺-dependent channel openings are voltage dependent. In the presence of micromolar luminal Ca²⁺, theP _o and duration of sulmazole-activated openings are greater atnegative voltages. However, at millimolar luminal [Ca²⁺], long openings are also observed at positive voltages and theP _o appears to be similar at positive and negative voltages. Our findings indicate thatthe regulation of channel gating by luminal Ca²⁺ depends on the mechanism of channel activation.We would like to thank Dr Allan Lindsay for the preparation of the purified SR Ca²⁺-release channels. This work was supported by the British Heart Foundation.     

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.     

Low-voltage triggering of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle cells

This study investigated the interaction between L-type Ca²⁺ current (ICaL) and Ca²⁺ release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Na_o⁺:K_I⁺) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca²⁺ current, TTX-sensitive ICa (ICaTTX), or slip mode Ca²⁺ conductance via INa⁺ were not responsible for low-voltage SRCR. However, L-type Ca²⁺ currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca²⁺ release from the SR at negative potentials is due to activation of L-type Ca²⁺ channels. heart; calcium current; low-voltage activation     

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca²⁺ release channel (ryanodine receptor) in excitable tissues. Using [³H]ryanodine, a 30 S protein complex comprised of four polypeptides of M_r 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [³H]ryanodine binding and Ca²⁺ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary The two high affinity calcium binding sites of the cardiac (Ca²⁺ + Mg²⁺)-ATPase have been identified with the use of Eu³⁺. Eu³⁺ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H₂O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca²⁺ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.     

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel.

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.     

Regulation of Ca2+ release from sarcoplasmic reticulum in skeletal muscles

Ca²⁺ release from skeletal sarcoplasmic reticulum (SR) could be regulated by at least three mechanisms: 1) Ca²⁺, 2) calmodulin, and 3) Ca²⁺/calmodulin-dependent phosphorylation. Bell-shaped Ca²⁺-dependence, of Ca²⁺ release from both actively- and passively-loaded SR vesicles suggest that opening and closing of the Ca²⁺ release channel could be regulated by [Ca²⁺ _o] . The time- and concentration-dependent inhibition of Ca ²⁺ release from skeletal SR by calmodulin was also studied using passively-Ca²⁺ loaded SR vesicles. Up to 50% of Ca ²⁺ release was inhibited by calmodulin (0.01–0.5 µM); this inhibition required 5–15 min preincubation time. The hypothesis that Ca²⁺/calmodulin-dependent phosphorylation of a 60 kDa protein regulates Ca²⁺ release from skeletal SR was tested by stopped-flow fluorometry using passively-Ca²⁺-loaded SR vesicles. Approximately 80% of the initial rates of Ca²⁺-induced Ca²⁺ release was inhibited by the phosphorylation within 2 min of incubation of the SR with Mg·ATP and calmodulin. We identified two types of 60 kDa phosphoproteins in the rabbit skeletal SR, which was distinguished by solubility of the protein in CHAPS. The CHAPS-soluble 60 kDa phosphoprotein was purified by column chromatography on DEAE-Sephacel, heparin-agarose, and hydroxylapatite. Analyses of the purified protein indicate that the CHAPS-soluble 60 kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding isoforms of PGM were cloned and sequenced using synthetic oligonucleotides. Two types of PGM isoforms (Type I and Type 11) were identified. The translated amino acid sequences show that Type II isoform is SR-form. Our results are significant in terms of understanding evidence of an association of glycolytic and glycogenolytic enzymes with SR and a role in the regulation of SR functions. (Mol Cell Biochem 114: 105-108, 1992)     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Effects of azumolene on doxorubicin-induced Ca2+ release from skeletal and cardiac muscle sarcoplasmic reticulum

The mechanism of doxorubicin-induced Ca2+ release from skeletal and cardiac muscle sarcoplasmic reticulum (SR) was studied by examining the effects of azumolene (a water soluble dantrolene analog) on doxorubicin-mediated Ca2+ release and ryanodine binding. Doxorubicin induced a rapid Ca2+ release from both skeletal and cardiac SR in a similar concentration range (EC50 = 5-10 microM). Maximal doxorubicin-induced Ca2+ release was seen at 2 and 0.2 microM Ca2+ for skeletal and cardiac SR, respectively. Addition of 400 microM azumolene caused approx. 30% inhibition of doxorubicin-induced Ca2+ release from both skeletal and cardiac SR; skeletal SR had significantly higher sensitivity to azumolene than cardiac SR. In the presence of Ca2+, doxorubicin increased [3H]ryanodine binding to both skeletal and cardiac SR; whereas in the absence of Ca2+, doxorubicin led to significant ryanodine binding to skeletal SR, but not to cardiac SR. In both types of SR, doxorubicin-activated, but not Ca2+ activated ryanodine binding was inhibited by azumolene. Azumolene sensitivity for inhibition of doxorubicin-activated ryanodine binding was much higher in skeletal SR than cardiac SR, consistent with the results for effects of azumolene on Ca2+ release. Our results are consistent with the possibility that azumolene inhibits doxorubicin binding by direct competition for the drug receptor(s).     

Reactive disulfide compounds induce Ca2+ release from cardiac sarcoplasmic reticulum

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to a disulfide bond, 2,2'dithiodipyridine (2,2' DTDP) and 4,4' dithiodipyridine (4,4' DTDP), induce Ca2+ release from isolated canine cardiac sarcoplasmic reticulum (SR) vesicles. RDSs are absolutely specific to free sulfhydryl (SH) groups and oxidize SH sites of low pKa via a thiol-disulfide exchange reaction, with the stoichiometric production of thiopyridone in the medium. As in skeletal SR, this reaction caused large increases in the Ca2+ permeability of cardiac SR and the number of SH sites oxidized by RDSs was kinetically and quantitatively measured through the absorption of thiopyridone. RDS-induced Ca2+ release from cardiac SR was characterized and compared to the action of RDSs on skeletal SR and to Ca2(+)-induced Ca2+ release. (i) RDS-induced Ca2+ release from cardiac SR was dependent on ionized Mg2+, with maximum rates of release occurring at 0.5 and 1 mM Mg2+free for 2,2' DTDP and 4,4' DTDP, respectively. (ii) In the presence of adenine nucleotides (0.1-1 mM), the oxidation of SH sites in cardiac SR by exogenously added RDS was inhibited, which, in turn, inhibited Ca2+ release induced by RDSs. (iii) Conversely, when the oxidation reaction between RDSs and cardiac SR was completed and Ca2+ release pathways were opened, subsequent additions of adenine nucleotides stimulated Ca2+ efflux induced by RDSs. (iv) Sulfhydryl reducing agents (e.g., dithiothreitol, DTT, 1-5 mM) inhibited RDS-induced Ca2+ efflux in a concentration-dependent manner. (v) RDSs elicited Ca2+ efflux from passively loaded cardiac SR vesicles (i.e., with nonfunctional Ca2+ pumps in the absence of Mg-ATP) and stimulated Ca2(+)-dependent ATPase activity, which indicated that RDS uncoupled Ca2+ uptake and did not act at the Ca2+, Mg2(+)-ATPase. These results indicate that RDSs selectively oxidize critical sulfhydryl site(s) on or adjacent to a Ca2+ release channel protein channel and thereby trigger Ca2+ release. Conversely, reduction of these sites reverses the effects of RDSs by closing Ca2+ release channels, which results in active Ca2+ reuptake by Ca2+, Mg2(+)-ATPase. These compounds can thus provide a method to covalently label and identify the protein involved in Ca2+ release from cardiac SR.     

Mechanism of calmodulin inhibition of cardiac sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor)

The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca(2+) concentrations. CaM reduced channel open probability at <10 micro M Ca(2+) by decreasing channel events and mean open times and increasing mean close times. At >10 micro M Ca(2+), CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to -50 mV and from -50 mV to +50 mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca(2+) fluxes. CaM decreased the duration of the open events after the voltage switch from -50 mV to +50 mV. In parallel experiments, a Ca(2+)-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca(2+) release.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.     

Structural and functional correlation of the trypsin-digested Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel.