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Rapid, simple and sensitive microassay for skeletal muscle homogenates in the functional assessment of the Ca-release channel of sarcoplasmic reticulum: Application to diagnosis of susceptibility to malignant hyperthermia

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Abstract

A microassay is demonstrated for functional characterization of the Ca2+-release channel (CRC) of sarcoplasmic reticulum (SR) of skeletal muscle using swine with susceptibility to malignant hyperthermia (MH). Diluted muscle homogenates, indo-1 and ratiometric dual-emission spectrofluorometry are used to monitor Ca2+-lowering activity in real-time in the presence and absence of ryanodine at exposures that open and close the CRC. Reactions are initiated with 50 µM CaCl2 to raise ionized Ca2+ concentration near 1 µM and MgATP to activate the Ca2+-ATPase pump. Oxalate is included to precipitate Ca2+ within the SR. The assay requires less than 30 mg muscle, which may be cryopreserved, and is completed within 20 min of thawing the tissue. Maximum SR Ca2+-ATPase pumping and CRC activities, degree of CRC activation, and Ca2+-buffering capacity can be determined. Using this assay we studied muscle from MH-susceptible swine and demonstrated that whereas maximal Ca2+-ATPase pumping and CRC activities are normal, the CRC activity after addition of a bolus of Ca2+ is 50% greater in heterozygotes and 100% greater in homozygotes for the MH mutation. Hypersensitivity to CRC agonists, such as caffeine, and an associated hyposensitivity to CRC antagonists such as Mg2+ is also demonstrated. Genotypes for the MH mutation site can be discriminated from each other by determining Ca2+-lowering activities and the effect of ryanodine on them. (Mol Cell Biochem 167: 61-72, 1997)

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Authors and Affiliations

  1. Human Safety Department, Miami Valley Laboratories, The Procter and Gamble Company, 11810 East Miami River Road, Ross, Ohio, 45081, USA

    P.J. O'Brien

  2. Department of Pharmacology and Toxicology, School of Medicine, 501 North Columbia Road, Grand Forks, North Dakota, 58202, USA

    G. Li

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  2. G. Li
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O'Brien, P., Li, G. Rapid, simple and sensitive microassay for skeletal muscle homogenates in the functional assessment of the Ca-release channel of sarcoplasmic reticulum: Application to diagnosis of susceptibility to malignant hyperthermia. Mol Cell Biochem 167, 61–72 (1997). https://doi.org/10.1023/A:1006867521140

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