Temperature and RyR1 Regulate the Activation Rate of Store-Operated Ca2+ Entry Current in Myotubes

AbstractStore-operated calcium entry (SOCE) is an important Ca2+ entry pathway in skeletal muscle. However, direct electrophysiological recording and full characterization of the underlying SOCE current in skeletal muscle cells (ISkCRAC) has not been reported. Here, we characterized the biophysical properties, pharmacological profile, and molecular identity of ISkCRAC in skeletal myotubes, as well as the regulation of its rate of activation by temperature and the type I ryanodine receptor (RyR1). ISkCRAC exhibited many hallmarks of Ca2+ release activated Ca2+ currents (ICRAC): store dependence, strong inward rectification, positive reversal potential, limited cesium permeability, and sensitivity to SOCE channel blockers. ISkCRAC was reduced by siRNA knockdown of stromal interaction molecule 1 and expression of dominant negative Orai1. Average ISkCRAC current density at −80mV was 1.00 ± 0.05 pA/pF. In the presence of 20 mM intracellular EGTA, ISkCRAC activation occurred over tens of seconds during repetitive depolarization at 0.5Hz and was inhibited by treatment with 100 μM ryanodine. The rate of SOCE activation was reduced threefold in myotubes from RyR1-null mice and increased 4.6-fold at physiological temperatures (35–37°C). These results show that ISkCRAC exhibits similar biophysical, pharmacological, and molecular properties as ICRAC in nonexcitable cells and its rate of activation during repetitive depolarization is strongly regulated by temperature and RyR1 activity

The Pore Region of the Skeletal Muscle Ryanodine Receptor Is a Primary Locus for Excitation-Contraction Uncoupling in Central Core Disease

Human central core disease (CCD) is caused by mutations/deletions in the gene that encodes the skeletal muscle ryanodine receptor (RyR1). Previous studies have shown that CCD mutations in the NH2-terminal region of RyR1 lead to the formation of leaky SR Ca2+ release channels when expressed in myotubes derived from RyR1-knockout (dyspedic) mice, whereas a COOH-terminal mutant (I4897T) results in channels that are not leaky to Ca2+ but lack depolarization-induced Ca2+ release (termed excitation-contraction [EC] uncoupling). We show here that store depletion resulting from NH2-terminal (Y523S) and COOH-terminal (Y4795C) leaky CCD mutant release channels is eliminated after incorporation of the I4897T mutation into the channel (Y523S/I4897T and Y4795C/I4897T). In spite of normal SR Ca2+ content, myotubes expressing the double mutants lacked voltage-gated Ca2+ release and thus exhibited an EC uncoupling phenotype similar to that of I4897T-expressing myotubes. We also show that dyspedic myotubes expressing each of seven recently identified CCD mutations located in exon 102 of the RyR1 gene (G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, R4913G) behave as EC-uncoupled release channels. Interestingly, voltage-gated Ca2+ release was nearly abolished (reduced ∼90%) while caffeine-induced Ca2+ release was only marginally reduced in R4892W-expressing myotubes, indicating that this mutation preferentially disrupts voltage-sensor activation of release. These data demonstrate that CCD mutations in exon 102 disrupt release channel permeation to Ca2+ during EC coupling and that this region represents a primary molecular locus for EC uncoupling in CCD