Involvement of the 60 kDa phosphoprotein in the regulation of Ca 2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

Abstract

Junctional sarcoplasmic reticulum (SR) vesicles isolated from back muscles of normal and malignant hyperthermia susceptible (MHS) pigs were phosphorylated by addition of MgATP in the presence of 5 mM Ca 2+ and 1 μM calmodulin (CaM). The major site of phosphorylation was a 60 kDa protein both in normal and MHS SR. The maximal amount of phosphorylation in MHS SR (5 pmol P/mg SR) was significantly lower than that in the normal SR (12 pmol P/mg SR). The phosphorylated 60 kDa protein was spontaneously dephosphorylated both in normal and MHS SR. Ca 2+ release from the passively loaded SR was induced by a Ca 2+-jump, and monitored by stopped-flow fluorometry using chlorotetracycline. In the absence of preincubation with MgATP, no significant difference was found in any of the kinetic parameters of Ca 2+ release between normal and MHS SR. Upon addition of 20 μM MgATP to the passively loaded SR to phosphorylate the 60 kDa protein, the initial rate of Ca 2+ release in normal SR significantly decreased from 659 ± 102to361 ± 105 nmol Ca 2+/mg SR per s, whereas in MHS SR the rate decreased from 749 ± 124 to 652 ± 179nmol Ca 2+/mg SR per s. Addition of 20 μM adenosine 5′-[β,γ-imido]triphosphate (p[NH]ppA) did not significantly alter the initial rate of Ca 2+ release both in normal and MHS SR. These results suggest that the previously reported higher Ca 2+ release rate in MHS SR (Kim et al. (1984) Biochim. Biophys. Acta 775, 320–327) is at least partly due to the reduced extent of the Ca 2+/CaM-dependent phosphorylation of the 60 kDa protein. Two-dimensional gel electrophoresis study showed that amount of a protein with M r = 55 000 was significantly lower in MHS SR than in normal SR suggesting that the abnormally lower amount of 55 kDa protein would cause the lower amount of phosphorylation of the 60 kDa protein in MHS SR.