American Journal of Physiology-cell Physiology, Jul 1, 1994
The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine r... more The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine receptor (DHPR), and SR ryanodine receptor (RyR) was determined in muscle of pigs homozygous for the normal RyR allele and homozygous or heterozygous for the malignant hyperthermia-susceptible (MHS) RyR allele. Total muscle membranes isolated from 1-day-old pigs of the three different genotypes did not differ in the content of any of these proteins. However, at 28 days of age, crude membranes and total muscle homogenates from homozygous MHS pigs exhibited only 61-81% of the [3H]PN 200-110 or [3H]ryanodine binding of identical preparations isolated from normal pigs; these MHS membranes also contained only 50% of the normal content of each of the DHPR subunits. The crude membranes and muscle homogenates from heterozygous pigs were intermediate to both types of homozygotes in terms of [3H]PN 200-110 binding, [3H]ryanodine binding, and the content of the DHPR subunits. However, membrane preparations enriched in triadic junctional proteins isolated from 3- to 4-mo-old pigs of the three different genotypes did not differ in their [3H]PN 200-110 binding, [3H]ryanodine binding, or Ca(2+)-ATPase activities. We conclude that, although the stoichiometry of the RyR to DHPR is not altered, the presence of the MHS RyR allele during muscle development results in a decreased relative content of these two proteins. This is probably due to a lower junctional membrane content and may be an important ultrastructural consequence of the altered sarcoplasmic Ca2+ regulation in MHS muscle.
Archives of Biochemistry and Biophysics, Apr 1, 1990
When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptib... more When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.
American Journal of Physiology-cell Physiology, Oct 1, 1989
Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to ... more Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to both in vivo and in vitro muscle responses to halothane (E. M. Gallant, J. R. Mickelson, B. D. Roggow, S. K. Donaldson, C. F. Louis, and W. E. Rempel. Am. J. Physiol. 257 (Cell Physiol. 26): C781-C786, 1989). In this paper heavy sarcoplasmic reticulum (SR) preparations were isolated from the muscles of pigs of all three genotypes. The rate of calcium release from SR of pigs homozygous for the halothane-sensitivity gene was approximately twice that of SR from pigs homozygous for the normal allele. Furthermore, in the presence of 6 microM Ca2+, the binding of [3H]ryanodine to SR isolated from the homozygous halothane-sensitive pigs was of a higher affinity than was the binding to SR isolated from the homozygous normal pigs (Kd = 70-90 vs. 265 nM, respectively). The SR from pigs heterozygous for the halothane-sensitivity gene, however, demonstrated intermediate values for the rate of calcium release and the affinity for [3H]ryanodine (Kd = 192 nM). Thus the alterations in heavy SR calcium release and [3H]ryanodine binding in the pigs containing one copy of the halothane-sensitivity gene demonstrate a distinct heterozygote phenotype. These data also suggest that the protein product of this gene is closely associated with, and perhaps identical to, the SR calcium release channel-ryanodine receptor protein.
Previous studies have demonstrated that skeletal muscle from individuals susceptible to malignant... more Previous studies have demonstrated that skeletal muscle from individuals susceptible to malignant hyperthermia (MH) has a defect associated with the mechanism of calcium release from its intracellular storage sites in the sarcoplasmic reticulum (SR). In this report we demonstrate that the [3H]ryanodine receptor of isolated MH-susceptible (MHS) porcine heavy SR exhibits an altered Ca2+ dependence of [3H]ryanodine binding at the low affinity Ca2+ site as well as a lower Kd for ryanodine (92 versus 265 nM) when compared to normal porcine SR. The Bmax of the normal and MHS [3H] ryanodine receptor (9.3-12.6 pmol/mg) was not significantly different, and analysis of MHS and normal SR proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis did not reveal a significant difference in the intensity of Coomassie Blue staining of the spanning protein/ryanodine receptor region of the gels (Mr greater than 300,000). We also find that MHS porcine muscle intact fiber bundles exhibit a 5-10-fold lower ryanodine threshold for twitch and tetanus inhibition, and contracture onset when compared to normal muscle. Since the SR ryanodine receptor is a calcium release channel as well as a component intimately involved in transverse tubule-SR communication, abnormalities in the skeletal muscle ryanodine receptor may be responsible for the abnormal SR calcium release and contractile properties demonstrated by MHS muscle.
Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to ... more Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to both in vivo and in vitro muscle responses to halothane (E. M. Gallant, J. R. Mickelson, B. D. Roggow, S. K. Donaldson, C. F. Louis, and W. E. Rempel. Am. J. Physiol. 257 (Cell Physiol. 26): C781-C786, 1989). In this paper heavy sarcoplasmic reticulum (SR) preparations were isolated from the muscles of pigs of all three genotypes. The rate of calcium release from SR of pigs homozygous for the halothane-sensitivity gene was approximately twice that of SR from pigs homozygous for the normal allele. Furthermore, in the presence of 6 microM Ca2+, the binding of [3H]ryanodine to SR isolated from the homozygous halothane-sensitive pigs was of a higher affinity than was the binding to SR isolated from the homozygous normal pigs (Kd = 70-90 vs. 265 nM, respectively). The SR from pigs heterozygous for the halothane-sensitivity gene, however, demonstrated intermediate values for the rate of calciu...
The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine r... more The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine receptor (DHPR), and SR ryanodine receptor (RyR) was determined in muscle of pigs homozygous for the normal RyR allele and homozygous or heterozygous for the malignant hyperthermia-susceptible (MHS) RyR allele. Total muscle membranes isolated from 1-day-old pigs of the three different genotypes did not differ in the content of any of these proteins. However, at 28 days of age, crude membranes and total muscle homogenates from homozygous MHS pigs exhibited only 61-81% of the [3H]PN 200-110 or [3H]ryanodine binding of identical preparations isolated from normal pigs; these MHS membranes also contained only 50% of the normal content of each of the DHPR subunits. The crude membranes and muscle homogenates from heterozygous pigs were intermediate to both types of homozygotes in terms of [3H]PN 200-110 binding, [3H]ryanodine binding, and the content of the DHPR subunits. However, membrane prepa...
Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele... more Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele demonstrate altered sarcoplasmic reticulum (SR) ryanodine binding and Ca2+ release channel regulatory properties when compared with normal pigs homozygous for the Arg615 allele. While solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified MHS and normal ryanodine receptors had a similar dissociation constant (Kd) for ryanodine, maximum binding, and Ca2+ concentration for half-maximal stimulation and inhibition of ryanodine binding (Ca2+(0.5)); however, after reconstitution into proteoliposomes, the purified MHS and normal receptors had Kd values for ryanodine of 75 and 150 nM, respectively, which were significantly different. The purified MHS and normal porcine ryanodine receptors also had similar single-channel Cs+ conductance, optimal cis-Ca2+ for channel opening, and cis-Ca2+(0.5) for channel activation. Significantly, at inactivating levels of cis-Ca2...
Journal of the Science of Food and Agriculture, 2009
BACKGROUND: In previous research, we demonstrated that cell wall polysaccharide (CWP) levels of s... more BACKGROUND: In previous research, we demonstrated that cell wall polysaccharide (CWP) levels of soybean (Glycine max L.) cotyledons are negatively correlated with the sum of seed oil and protein content. Although the results suggest that reducing cotyledon CWP levels would be desirable, it is not known whether CWP are mobilized during early seedling growth and, if so, to what extent mobilization contributes to seed reserves.RESULTS: Ungerminated (dry) seeds contained equivalent amounts [approximately 20 mg (cotyledon pair)−1] of non‐cell wall carbohydrates (NCWC) and CWP. Galactose and arabinose accounted for 47% of total CWP in cotyledons of dry seeds. Measured 14 days after planting (DAP), the levels of NCWC and CWP were reduced 98% and 34%, respectively, in cotyledons of seedlings grown under a 16‐h photoperiod. Measured 14 DAP, greater than 85% of cotyledon cell wall galactose plus arabinose was mobilized. The transformation of the cotyledon to a photosynthetic organ was associa...
The skeletal muscle ryanodine receptor of malignant hyperthermia‐susceptible (MHS) pigs contains ... more The skeletal muscle ryanodine receptor of malignant hyperthermia‐susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca2+ channel activity. In isolated SR membranes the Arg615 to Cys615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg615 cleavage site. Furthermore, trypsin treatment released 86–99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86–99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca2+ channel regulatory sites.
When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptib... more When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.
American Journal of Physiology-cell Physiology, Jul 1, 1994
The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine r... more The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine receptor (DHPR), and SR ryanodine receptor (RyR) was determined in muscle of pigs homozygous for the normal RyR allele and homozygous or heterozygous for the malignant hyperthermia-susceptible (MHS) RyR allele. Total muscle membranes isolated from 1-day-old pigs of the three different genotypes did not differ in the content of any of these proteins. However, at 28 days of age, crude membranes and total muscle homogenates from homozygous MHS pigs exhibited only 61-81% of the [3H]PN 200-110 or [3H]ryanodine binding of identical preparations isolated from normal pigs; these MHS membranes also contained only 50% of the normal content of each of the DHPR subunits. The crude membranes and muscle homogenates from heterozygous pigs were intermediate to both types of homozygotes in terms of [3H]PN 200-110 binding, [3H]ryanodine binding, and the content of the DHPR subunits. However, membrane preparations enriched in triadic junctional proteins isolated from 3- to 4-mo-old pigs of the three different genotypes did not differ in their [3H]PN 200-110 binding, [3H]ryanodine binding, or Ca(2+)-ATPase activities. We conclude that, although the stoichiometry of the RyR to DHPR is not altered, the presence of the MHS RyR allele during muscle development results in a decreased relative content of these two proteins. This is probably due to a lower junctional membrane content and may be an important ultrastructural consequence of the altered sarcoplasmic Ca2+ regulation in MHS muscle.
Archives of Biochemistry and Biophysics, Apr 1, 1990
When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptib... more When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.
American Journal of Physiology-cell Physiology, Oct 1, 1989
Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to ... more Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to both in vivo and in vitro muscle responses to halothane (E. M. Gallant, J. R. Mickelson, B. D. Roggow, S. K. Donaldson, C. F. Louis, and W. E. Rempel. Am. J. Physiol. 257 (Cell Physiol. 26): C781-C786, 1989). In this paper heavy sarcoplasmic reticulum (SR) preparations were isolated from the muscles of pigs of all three genotypes. The rate of calcium release from SR of pigs homozygous for the halothane-sensitivity gene was approximately twice that of SR from pigs homozygous for the normal allele. Furthermore, in the presence of 6 microM Ca2+, the binding of [3H]ryanodine to SR isolated from the homozygous halothane-sensitive pigs was of a higher affinity than was the binding to SR isolated from the homozygous normal pigs (Kd = 70-90 vs. 265 nM, respectively). The SR from pigs heterozygous for the halothane-sensitivity gene, however, demonstrated intermediate values for the rate of calcium release and the affinity for [3H]ryanodine (Kd = 192 nM). Thus the alterations in heavy SR calcium release and [3H]ryanodine binding in the pigs containing one copy of the halothane-sensitivity gene demonstrate a distinct heterozygote phenotype. These data also suggest that the protein product of this gene is closely associated with, and perhaps identical to, the SR calcium release channel-ryanodine receptor protein.
Previous studies have demonstrated that skeletal muscle from individuals susceptible to malignant... more Previous studies have demonstrated that skeletal muscle from individuals susceptible to malignant hyperthermia (MH) has a defect associated with the mechanism of calcium release from its intracellular storage sites in the sarcoplasmic reticulum (SR). In this report we demonstrate that the [3H]ryanodine receptor of isolated MH-susceptible (MHS) porcine heavy SR exhibits an altered Ca2+ dependence of [3H]ryanodine binding at the low affinity Ca2+ site as well as a lower Kd for ryanodine (92 versus 265 nM) when compared to normal porcine SR. The Bmax of the normal and MHS [3H] ryanodine receptor (9.3-12.6 pmol/mg) was not significantly different, and analysis of MHS and normal SR proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis did not reveal a significant difference in the intensity of Coomassie Blue staining of the spanning protein/ryanodine receptor region of the gels (Mr greater than 300,000). We also find that MHS porcine muscle intact fiber bundles exhibit a 5-10-fold lower ryanodine threshold for twitch and tetanus inhibition, and contracture onset when compared to normal muscle. Since the SR ryanodine receptor is a calcium release channel as well as a component intimately involved in transverse tubule-SR communication, abnormalities in the skeletal muscle ryanodine receptor may be responsible for the abnormal SR calcium release and contractile properties demonstrated by MHS muscle.
Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to ... more Pigs heterozygous for the halothane-sensitivity gene exhibit a distinct phenotype with regard to both in vivo and in vitro muscle responses to halothane (E. M. Gallant, J. R. Mickelson, B. D. Roggow, S. K. Donaldson, C. F. Louis, and W. E. Rempel. Am. J. Physiol. 257 (Cell Physiol. 26): C781-C786, 1989). In this paper heavy sarcoplasmic reticulum (SR) preparations were isolated from the muscles of pigs of all three genotypes. The rate of calcium release from SR of pigs homozygous for the halothane-sensitivity gene was approximately twice that of SR from pigs homozygous for the normal allele. Furthermore, in the presence of 6 microM Ca2+, the binding of [3H]ryanodine to SR isolated from the homozygous halothane-sensitive pigs was of a higher affinity than was the binding to SR isolated from the homozygous normal pigs (Kd = 70-90 vs. 265 nM, respectively). The SR from pigs heterozygous for the halothane-sensitivity gene, however, demonstrated intermediate values for the rate of calciu...
The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine r... more The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine receptor (DHPR), and SR ryanodine receptor (RyR) was determined in muscle of pigs homozygous for the normal RyR allele and homozygous or heterozygous for the malignant hyperthermia-susceptible (MHS) RyR allele. Total muscle membranes isolated from 1-day-old pigs of the three different genotypes did not differ in the content of any of these proteins. However, at 28 days of age, crude membranes and total muscle homogenates from homozygous MHS pigs exhibited only 61-81% of the [3H]PN 200-110 or [3H]ryanodine binding of identical preparations isolated from normal pigs; these MHS membranes also contained only 50% of the normal content of each of the DHPR subunits. The crude membranes and muscle homogenates from heterozygous pigs were intermediate to both types of homozygotes in terms of [3H]PN 200-110 binding, [3H]ryanodine binding, and the content of the DHPR subunits. However, membrane prepa...
Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele... more Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele demonstrate altered sarcoplasmic reticulum (SR) ryanodine binding and Ca2+ release channel regulatory properties when compared with normal pigs homozygous for the Arg615 allele. While solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified MHS and normal ryanodine receptors had a similar dissociation constant (Kd) for ryanodine, maximum binding, and Ca2+ concentration for half-maximal stimulation and inhibition of ryanodine binding (Ca2+(0.5)); however, after reconstitution into proteoliposomes, the purified MHS and normal receptors had Kd values for ryanodine of 75 and 150 nM, respectively, which were significantly different. The purified MHS and normal porcine ryanodine receptors also had similar single-channel Cs+ conductance, optimal cis-Ca2+ for channel opening, and cis-Ca2+(0.5) for channel activation. Significantly, at inactivating levels of cis-Ca2...
Journal of the Science of Food and Agriculture, 2009
BACKGROUND: In previous research, we demonstrated that cell wall polysaccharide (CWP) levels of s... more BACKGROUND: In previous research, we demonstrated that cell wall polysaccharide (CWP) levels of soybean (Glycine max L.) cotyledons are negatively correlated with the sum of seed oil and protein content. Although the results suggest that reducing cotyledon CWP levels would be desirable, it is not known whether CWP are mobilized during early seedling growth and, if so, to what extent mobilization contributes to seed reserves.RESULTS: Ungerminated (dry) seeds contained equivalent amounts [approximately 20 mg (cotyledon pair)−1] of non‐cell wall carbohydrates (NCWC) and CWP. Galactose and arabinose accounted for 47% of total CWP in cotyledons of dry seeds. Measured 14 days after planting (DAP), the levels of NCWC and CWP were reduced 98% and 34%, respectively, in cotyledons of seedlings grown under a 16‐h photoperiod. Measured 14 DAP, greater than 85% of cotyledon cell wall galactose plus arabinose was mobilized. The transformation of the cotyledon to a photosynthetic organ was associa...
The skeletal muscle ryanodine receptor of malignant hyperthermia‐susceptible (MHS) pigs contains ... more The skeletal muscle ryanodine receptor of malignant hyperthermia‐susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca2+ channel activity. In isolated SR membranes the Arg615 to Cys615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg615 cleavage site. Furthermore, trypsin treatment released 86–99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86–99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca2+ channel regulatory sites.
When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptib... more When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.
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