Caffeine sensitivity of native RyR channels from normal and malignant hyperthermic pigs: Effects of a DHPR II-III loop peptide

Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine sensitivity of skeletal muscle contraction and Ca²⁺ release from the sarcoplasmic reticulum is enhanced, but surprisingly, the caffeine sensitivity of purified porcine ryanodine receptor Ca²⁺-release channels (RyRs) is not affected by the MH mutation (Arg⁶¹⁵Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins) were activated by caffeine at 100- to 1,000-fold lower concentrations than native normal pig RyRs. In addition, the results show that the mutant ryanodine receptor channels were less sensitive to high-affinity activation by a peptide (Cs) that corresponds to a part of the II-III loop of the skeletal dihydropyridine receptor (DHPR). Furthermore, subactivating concentrations of peptide Cs enhanced the response of normal pig and rabbit RyRs to caffeine. In contrast, the caffeine sensitivity of MH RyRs was not enhanced by the peptide. These novel results showed that in MH-susceptible pig muscles 1) the caffeine sensitivity of native RyRs was enhanced, 2) the sensitivity of RyRs to a skeletal II-III loop peptide was depressed, and 3) an interaction between the caffeine and peptide Cs activation mechanisms seen in normal RyRs was lost.