Arg⁶¹⁵cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca²⁺ release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr⁶⁷¹-Leu⁶⁹⁰; 36 μM) enhanced the rate of Ca²⁺ release from normal SR (SRN) and from SR of MH-susceptible muscle (SRMH) by 10 ± 3.2 nmole/mg/min and 76 ± 9.7 nmole/mg/min, respectively. Ca²⁺ release from SR_N or SR_MH was not increased by control peptide NB (Gly⁶⁸⁹-Lys⁷⁰⁸). AS (scrambled A sequence; 36 μM) did not alter Ca²⁺ release from SR_N, but increased release from SR_MH by 29 ± 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR_MH) were up to about fourfold more strongly activated by peptide A ≥1 nM) than normal RyR channels (RyRN) at -40 mV. Neither NB or AS activated RyR_N. RyR_MH showed an ∼ 1.8-fold increase in mean current with 30 μM AS. Inhibition at +40 mV was stronger in RyR_MH and seen with peptide A (≥0.6 μM) and AS (≥0.6 μM), but not NB. These results show that the Arg⁶¹⁵Cys substitution in RyR_MH has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca²⁺ release from SR in MH-susceptible muscle.