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.
A microassay is demonstrated for functional characterization of the Ca(2+)-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 Ca(2+)-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 microM CaCl2 to raise ionized Ca2+ concentration near 1 microM and MgATP to activate the Ca(2+)-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 Ca(2+)-ATPase pumping and CRC activities, degree of CRC activation, and Ca(2+)-buffering capacity can be determined. Using this assay we studied muscle from MH-susceptible swine and demonstrated that whereas maximal Ca(2+)-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 Ca(2+)-lowering activities and the effect of ryanodine on them.
O'Brien
P J
PJ
Human Safety Department, Miami Valley Laboratories, Procter and Gamble Company, Ross, Ohio 45081, USA.
Li
G
G
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000378:metabolism; D004195:Disease Models, Animal; D004198:Disease Susceptibility; D005470:Fluorometry / Q000379:methods*; D006579:Heterozygote; D008274:Magnesium / Q000494:pharmacology; D008305:Malignant Hyperthermia / Q000175:diagnosis* / Q000235:genetics; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013552:Swine
D015220:Calcium Channels; D012433:Ryanodine; D002110:Caffeine; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium