BTP2 negatively regulates Orai1 and ryanodine receptor function in skeletal muscle

Background. BTP2 is known to block Orai1, the Ca2+ channel of store-operated Ca2+ entry (SOCE) but no detailed analysis has been undertaken in skeletal muscle, where the drug has been used extensively to study SOCE. Methodology. We trapped a Ca2+ sensitive dye in the tubular (t-) system of mechanically skinned fibres from rat to define the effect of BTP2 on SOCE in skeletal muscle fibres and used a cytoplasmic rhod-2 to track Ca2+ release in the presence of BTP2. Results. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of a resting Ca2+ conductance, likely to be through the Orai1 channel. Intriguingly, increasing concentrations of BTP2 displayed a hormetic effect on resting [Ca2+] in the t-system ([Ca2+]t−sys), shifting from inducing an accumulation of Ca2+ in the t-system presumably due to Orai1 channels blocking, to reducing the resting [Ca2+]t−sys. In absence of functional ryanodine receptors (RyRs), this biphasic effect was not observed, suggesting that above the hormetic zone, BTP2 impairs RyR function. Additionally, we found that BTP2 impairs the cytoplasmic Ca2+ transients during repetitive excitation-contraction coupling (EC coupling) cycles, independently of extracellular Ca2+ entry impairment. We determined that the release of Ca2+ through the RyR was inhibited by BTP2, strongly suggesting that the RyR was the point of inhibition during the cycles of EC coupling. Finally, we found that BTP2 inhibition of RyR-mediated Ca2+ release was independent of extracellular or intracellular application of the agent, indicating that BTP2 can impair RyR function in intact muscle. Conclusion. Our results show that both Ca2+ channels, the Orai1 and RyR, are negatively regulated by BTP2, shedding new light on previous work that applied BTP2 to block SOCE in muscle.