Stimulation and inhibition of [ 3H]ryanodine binding to sarcoplasmic reticulum from malignant hyperthermia susceptible pigs

Abstract

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 μ m Ca 2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg 2+) 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 m m ATP ( K d = 6.0 nM) or 10 m m caffeine ( K d = 28 nM) was significantly greater than that of the normal SR ( K d = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively); the B max (12–16 pmol/mg) was similar in all cases. The Ca 0.5 2+ for inhibition of [ 3H]ryanodine binding in the presence of 5 m m AMPPNP (238 vs 74 μ m for MHS and normal SR, respectively) and the Ca 0.5 2+ for stimulation of [[su3H]ryanodine binding in the presence of 5 m m caffeine (0.049 vs 0.070 μ m for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca 2+, MHS SR [ 3H]ryanodine binding was more sensitive to caffeine stimulation ( C 0.5 of 1.7 vs 3.4 m m) and was less sensitive to ruthenium red ( C 0.5 of 1.9 vs 1.2 μ m) or Mg 2+ inhibition ( C 0.5 of 0.34 vs 0.21 m m) 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.