Skeletal Muscle Ryanodine Receptor Research Articles

Direct Detection of Calmodulin Tuning by Ryanodine Receptor Channel Targets Using a Ca2+-Sensitive Acrylodan-Labeled Calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).

Mutational Analysis of Putative Calcium Binding Motifs within the Skeletal Ryanodine Receptor Isoform, RyR1

The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila

A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM.

Magnetic resonance imaging of muscle in congenital myopathies associated with RYR1 mutations

Mutations in the skeletal muscle ryanodine receptor ( RYR1) gene are associated with a wide range of phenotypes, comprising central core disease and distinct subgroups of multi-minicore disease. We report muscle MRI findings of 11 patients from eight families with RYR1 mutations ( n=9) or confirmed linkage to the RYR1 locus ( n=2). Patients had clinical features of a congenital myopathy with a wide variety of associated histopathological changes. Muscle MR images showed a consistent pattern characterized by (a) within the thigh: selective involvement of vasti, sartorius, adductor magnus and relative sparing of rectus, gracilis and adductor longus; (b) within the lower leg: selective involvement of soleus, gastrocnemii and peroneal group and relative sparing of the tibialis anterior. Our findings indicate that patients with RYR1-related congenital myopathies have a recognizable pattern of muscle involvement irrespective of the variability of associated histopathological findings. Muscle MRI may supplement clinical assessment and aid selection of genetic tests particularly in patients with non-diagnostic or equivocal histopathological features.

Intracellular Ca 2+ dynamics in malignant hyperthermia and central core disease: established concepts, new cellular mechanisms involved

Malignant hyperthermia (MH) and central core disease (CCD) are inherited human disorders of skeletal muscle Ca 2+ homeostasis. Both MH and CCD are linked to mutations and/or deletions in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the intracellular Ca 2+ release channel, which is essential to excitation–contraction (EC) coupling. Our knowledge on how mutations in RyR1 disrupt intracellular Ca 2+ homeostasis and EC coupling, eventually leading to MH and CCD has been recently improved, thanks to multidisciplinary studies ranging from clinical, single channel recordings, patch-clamp experiments, and molecular biology. This review presents a brief historical perspective, on how pioneer studies resulted in associating MH and CCD to RyR1. The review is also focused on discussing novel results in regard to pathophysiological consequences of specific MH/CCD RyR1 mutant proteins, which are representative of the different cellular mechanisms that are linked to either phenotype.

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

Different Regions in Skeletal and Cardiac Muscle Ryanodine Receptors Are Involved in Transducing the Functional Effects of Calmodulin

Calmodulin (CaM) inhibits the skeletal muscle ryanodine receptor-1 (RyR1) and cardiac muscle RyR2 at micromolar Ca(2+) but activates RyR1 and inhibits RyR2 at submicromolar Ca(2+) by binding to a single, highly conserved CaM-binding site. To identify regions responsible for the differential regulation of RyR1 and RyR2 by CaM, we generated chimeras encompassing and flanking the CaM-binding domain. We found that the exchange of the N- and C-terminal flanking regions differentially affected RyR1 and RyR2. A RyR1/RyR2 chimera with an N-terminal flanking RyR2 substitution (RyR2 amino acid (aa) 3537-3579) was activated by CaM in single channel measurements at both submicromolar and micromolar Ca(2+). A RyR2/RyR1 chimera with a C-terminal flanking the 86-amino acid RyR1 substitution (RyR1 aa 3640-3725) bound (35)S-CaM but was not inhibited by CaM at submicromolar Ca(2+). In this region, five non-conserved amino acid residues (RyR1 aa 3680 and 3682-3685 and RyR2 aa 3647 and 3649-3652) differentially affect RyR helical probability. Substitution of the five amino acid residues in RyR1 with those of RyR2 showed responses to CaM comparable with wild type RyR1. In contrast, substitution of the five amino acid residues in RyR2 with those of RyR1 showed loss of CaM inhibition, whereas substitution of the five RyR2 sequence residues in the RyR2 chimera containing the RyR1 calmodulin-binding domain and C-flanking sequence restored wild type RyR2 inhibition by CaM at submicromolar Ca(2+). The results suggest that different regions are involved in CaM modulation of RyR1 and RyR2. They further suggest that five non-conserved amino acids in the C-terminal region flanking the CaM-binding domain have a key role in CaM inhibition of RyR2.

Dantrolene Use in 3,4-Methylenedioxymethamphetamine (“Ecstasy”)–Mediated Hyperthermia

We thank Dr. Rusyniak et al. for their critical comments on our study about the induction of malignant hyperthermia (MH) in susceptible swine by 3,4-methylenedioxymethamphetamine (MDMA) (“ecstasy”).1However, some of the criticisms of our study must be relativized.First, to our knowledge, this is the first controlled study investigating the association between MDMA-induced hypermetabolic syndrome and MH. MH crisis is an acute clinical complication; therefore, the experimental setting for this study was following the clinical situation, and diagnosis of MH in our experiment could only be based on clinical parameters. The definition of the clinical cutoff parameters for MH crisis in our study was following the recommendations for clinical diagnosis of human MH crisis and previous animal studies. Increasing doses of MDMA induced a hypermetabolic state in MH-susceptible (MHS) as well as MH-normal (MHN) swine. However, the changes in the MHN swine after receiving a higher dose of MDMA (12 mg/kg) were moderate compared with the changes in MHS swine after 8 mg/kg MDMA, and all MHS swine fulfilled the defined criteria for MH.The only known differentiation between MHS and MHN swine is the presence of the Arg615-Cys point mutation on chromosome 6 leading to a functional impairment of the skeletal muscle ryanodine receptor (RyR1). We share the opinion of Dr. Rusyniak et al. that MDMA-induced hypermetabolism is not solely mediated through RyR1 complexes. However, the different reactions of MHS and MHN swine in our study are an indirect hint for activation of RyR1 after in vivo MDMA administration. The current study was aimed to prove whether MDMA is capable of inducing an MH syndrome, not to clarify the exact pathomechanism, i.e. , a possible mediation via the RyR1. Whether the RyR1 activation could be attributed to a direct effect of MDMA at the skeletal muscle or to a secondary effect of central stimulation, hyperthermia, or an MDMA-metabolite must therefore be clarified in future studies.The definition of an MH “trigger” is not as clear as mentioned in the letter of Dr. Rusyniak et al. From a clinical point of view, an MH trigger is a substance that is able to induce an MH crisis in a genetically determined individual in a clinically relevant dosage without any relevant cofactors. Following this definition, MDMA triggered MH in MHS swine in our study. We agree that cumulative intravenous administration of MDMA is not the common method of MDMA abuse. However, this course of action and measurement of corresponding MDMA plasma concentrations allowed us to determine a dose response and to underline the clinical relevance.The therapeutic regimen of MDMA-induced MH in our study was based on the standard clinical therapy of MH. Standardized therapy of MH in the MHS swine performed with dantrolene, sodium bicarbonate, and hyperventilation partly removed the clinical signs of MH immediately. The body temperature of the swine remained unchanged 15 min after therapy induction. We agree that the short observation time without the possibility to detect changes in body temperature was a weakness in our study design.Whether administration of dantrolene is useful in all patients with MDMA-induced hyperthermia could not be answered by our study. However, in a life-threatening clinical situation, “simple hyperthermia” could not be distinguished from “true malignant hyperthermia.” Therefore, in our opinion, dantrolene might be a lifesaving therapy option, and consequently, administration of dantrolene should be considered with respect to patient safety in cases of MDMA-mediated hyperthermic syndrome.* University Hospital Hamburg-Eppendorf, Hamburg, Germany. fiege@uke.uni-hamburg.de

Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling.

Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) alpha(1S)-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of alpha(1S) (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (alpha(1S) null) myotubes. Compared with wild-type alpha(1S), caffeine-activated Ca(2+) release occurred at approximately fivefold lower concentrations in nonexpressing and R1086H-expressing myotubes. Although maximal voltage-gated Ca(2+) release was similar in alpha(1S)- and R1086H-expressing myotubes, the voltage dependence of Ca(2+) release was shifted approximately 5 mV to more negative potentials in R1086H-expressing myotubes. Our results demonstrate that alpha(1S) functions as a negative allosteric modulator of release channel activation by caffeine/voltage and that the R1086H MH mutation in the intracellular III-IV linker disrupts this negative regulatory influence. Moreover, a low caffeine concentration (2 mM) caused a similar shift in voltage dependence of Ca(2+) release in alpha(1S)- and R1086H-expressing myotubes. Compared with alpha(1S)-expressing myotubes, maximal L channel conductance (G(max)) was reduced in R1086H-expressing myotubes (alpha(1S) 130 +/- 10.2, R1086H 88 +/- 6.8 nS/nF; P < 0.05). The decrease in G(max) did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio (G(max)/Q(max)) was similar in alpha(1S)- and R1086H-expressing myotubes and a similar decrease in G(max) was observed for an analogous mutation engineered into the cardiac L channel (R1217H). In addition, both R1086H and R1217H DHPRs targeted normally and colocalized with RyR1 in sarcoplasmic reticulum (SR)-sarcolemmal junctions. These results indicate that the R1086H MH mutation in alpha(1S) enhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators.

Functional characterization of malignant hyperthermia-associated RyR1 mutations in exon 44, using the human myotube model

Malignant hyperthermia (MH) is a pharmacogenetic disorder with an autosomal dominant inheritance. During exposure to triggering agents as volatile anaesthetics, affected individuals may develop a potentially fatal hypermetabolic syndrome caused by excessive calcium release from the sarcoplasmic reticulum in skeletal muscle. More than 60 MH associated mutations were found in the gene of skeletal muscle ryanodine receptor (RyR1), but only some of them have been functionally characterized. Primary human myotubes were cultured from carriers of RyR1 mutations in exon 44 (Ala2350Thr, Arg2355Trp, Gly2375Ala) and from MH non-susceptible individuals. Investigation of calcium homeostasis with the calcium sensitive probe Fura 2 showed a higher sensitivity to the ryanodine receptor agonists 4-chloro- m-cresol, caffeine and halothane for the myotubes derived from the mutation carriers as compared to those of the control group. The presence of RyR1 mutations with impact on calcium homeostasis emphasizes the functional significance of exon 44.

Selenium compounds modulate the calcium release channel/ryanodine receptor of rabbit skeletal muscle by oxidizing functional thiols

Selenium compounds, such as sodium selenite and Ebselen were shown to increase high affinity ryanodine binding to the skeletal muscle type ryanodine receptor (RyR1) at nanomolar concentrations, and inhibit the receptor at low micromolar concentrations. This biphasic response was observed in both concentration and time-dependent assays. Extensive washing did not reverse either the stimulation or suppression of receptor binding, but both were prevented or reversed by addition of reduced glutathione, GSH. Selenium compounds were also shown to induce Ca 2+ release from the isolated sarcoplasmic reticulum vesicles. Sodium selenite and Ebselen stimulated the skeletal muscle ryanodine receptor by oxidizing 14 of 47 free thiols per monomer on RyR1 (as detected with the alkylating agent 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin) (CPM). Oxidation of the remaining thiols by these selenium compounds resulted in inhibition of the ryanodine receptor.

Identification of two $alpha;RYR alleles and characterization of $alpha;RYR transcript variants in turkey skeletal muscle

We hypothesized that a mutation in the turkey skeletal muscle ryanodine receptor α isoform (αRYR) underlies turkey meat quality problems which are strikingly similar to pale, soft, exudative (PSE) pork. RT-PCR analysis of turkey αRYR mRNA covering amino acids 376 to 615 (numbered according to the human sequence) revealed at least three transcript variants. One transcript was homologous to the mammalian skeletal muscle RYR1 sequence in this region. The second transcript variant (AS-81) was characterized by the absence of 81 bases located at the beginning of exon 13, while the third transcript variant (AS-193) carried a deletion of 193 bases, corresponding to the entire exon 13. Two αRYR genomic DNA alleles (αRYR-I and αRYR-II) carrying the region of deletions in the turkey cDNA sequences were identified. Nucleotide sequence analysis demonstrated that the two alleles are identical in exon sequences but different in intron sequences. Comparison of genomic and cDNA sequences indicated that both AS-81 and AS-193 transcript variants probably arose via alternative splicing. Consistent with this mechanism, the last eight nucleotides of the 81 bases form a consensus sequence for a splice acceptor site. Both alleles could give rise to the AS-81 and AS-193 transcript variants via alternative splicing. Birds homozygous for αRYR-II tended to have superior meat quality indicators including significantly higher muscle pH at 15-min post mortem and lower muscle exudate at 24-h post mortem, compared to birds homozygous for αRYR-I.

Identification of two alphaRYR alleles and characterization of alphaRYR transcript variants in turkey skeletal muscle.

We hypothesized that a mutation in the turkey skeletal muscle ryanodine receptor alpha isoform (alphaRYR) underlies turkey meat quality problems which are strikingly similar to pale, soft, exudative (PSE) pork. RT-PCR analysis of turkey alphaRYR mRNA covering amino acids 376 to 615 (numbered according to the human sequence) revealed at least three transcript variants. One transcript was homologous to the mammalian skeletal muscle RYR1 sequence in this region. The second transcript variant (AS-81) was characterized by the absence of 81 bases located at the beginning of exon 13, while the third transcript variant (AS-193) carried a deletion of 193 bases, corresponding to the entire exon 13. Two alphaRYR genomic DNA alleles (alphaRYR-I and alphaRYR-II) carrying the region of deletions in the turkey cDNA sequences were identified. Nucleotide sequence analysis demonstrated that the two alleles are identical in exon sequences but different in intron sequences. Comparison of genomic and cDNA sequences indicated that both AS-81 and AS-193 transcript variants probably arose via alternative splicing. Consistent with this mechanism, the last eight nucleotides of the 81 bases form a consensus sequence for a splice acceptor site. Both alleles could give rise to the AS-81 and AS-193 transcript variants via alternative splicing. Birds homozygous for alphaRYR-II tended to have superior meat quality indicators including significantly higher muscle pH at 15-min post mortem and lower muscle exudate at 24-h post mortem, compared to birds homozygous for alphaRYR-I.

Phosphorylation of Ryanodine Receptors

Both cardiac and skeletal muscle ryanodine receptors (RyRs) are parts of large complexes that include a number of kinases and phosphatases. These RyRs have several potential phosphorylation sites in their cytoplasmic domains, but the functional consequences of phosphorylation and the identity of the enzymes responsible have been subjects of considerable controversy. Hyperphosphorylation of Ser-2809 in RyR2 (cardiac isoform) and Ser-2843 in RyR1 (skeletal isoform) has been suggested to cause the dissociation of the FK506-binding protein (FKBP) from RyRs, producing "leaky channels," but some laboratories find no relationship between phosphorylation and FKBP binding. Also debated is the identity of the kinases that phosphorylate these serines: cAMP-dependent protein kinase (PKA) versus calmodulin kinase II (CaMKII). Phosphorylation of other targets of these kinases could also alter calcium homeostasis. For example, PKA also phosphorylates phospholamban (PLB), altering the Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity. This review summarizes the major findings and controversies associated with phosphorylation of RyRs.

Different Regions in Skeletal and Cardiac Muscle Ryanodine Receptors Are Involved in Transducing the Functional Effects of Calmodulin

Different Regions in Skeletal and Cardiac Muscle Ryanodine Receptors Are Involved in Transducing the Functional Effects of Calmodulin

Oligomerization of the cardiac ryanodine receptor C-terminal tail.

The C-terminal 100 amino acids of the RyR (ryanodine receptor), referred to as the C-terminal tail, is a highly conserved sequence that is present in all known RyR isoforms and which has been implicated in channel function. Deleting the final 15 amino acids from the full-length skeletal muscle RyR resulted in an inactive channel, attributed to impaired assembly of a tetrameric RyR complex [Gao, Tripathy, Lu and Meissner (1997) FEBS Lett. 412, 223-226]. To account for these observations, the C-terminal tail itself may be an important molecular determinant of oligomerization. Alternatively, the large N-terminal cytoplasmic domain may fold back upon itself to interact with the C-terminal tail to provide a correctly folded tetrameric structure. We explored these possibilities for RyR2 (cardiac RyR) using the yeast two-hybrid interaction assay and in vitro translation followed by immunoprecipitation and chemical cross-linking. The data indicate that the C-terminal tail of RyR2 is capable of self-tetramerization. Moreover, a truncated C-terminal tail, lacking the final 15 amino acids, failed to self-associate. These observations suggest that the intrinsic ability of the RyR C-terminal tail to self-tetramerize may be vitally important for the oligomeric assembly of the native RyR channel.

Characterization of Recombinant Skeletal Muscle (Ser-2843) and Cardiac Muscle (Ser-2809) Ryanodine Receptor Phosphorylation Mutants

Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.

Stoichiometric Phosphorylation of Cardiac Ryanodine Receptor on Serine 2809 by Calmodulin-dependent Kinase II and Protein Kinase A

Stoichiometric Phosphorylation of Cardiac Ryanodine Receptor on Serine 2809 by Calmodulin-dependent Kinase II and Protein Kinase A

Unitary Ca2+ current through mammalian cardiac and amphibian skeletal muscle ryanodine receptor Channels under near-physiological ionic conditions.

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1–30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (∼55%) in mammalian and amphibian channels. Two amplitudes, differing by ∼35%, were found in amphibian channel studies, probably corresponding to α and β RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (∼40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.

Critical Amino Acid Residues Determine the Binding Affinity and the Ca2+ Release Efficacy of Maurocalcine in Skeletal Muscle Cells

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [3H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC50 of 12 nm. MCa decreases the sensitivity of [3H]ryanodine binding to inhibitory high Ca2+ concentrations and increases it to the stimulatory low Ca2+ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca2+ efflux as demonstrated by MCa effects on Ca2+ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca2+ increase due to Ca2+ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg24 for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [3H]ryanodine binding although with a decreased EC50 and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca2+ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca2+ release from SR.