BTP2 negatively regulates Orai1 and ryanodine receptor function in skeletal muscle

Abstract Background. BTP2 is known to block Orai1, the Ca 2+ channel of store-operated Ca 2+ 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 Ca 2+ 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 Ca 2+ release in the presence of BTP2. Results. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of Orai1 channel resting Ca 2+ conductance. Intriguingly, increasing concentrations of BTP2 displayed a hormetic effect on resting [Ca 2+ ] in the t-system ([Ca 2+ ] t-sys ), shifting from inducing an accumulation of Ca 2+ in the t-system presumably due to Orai1 channels blocking, to reducing the resting [Ca 2+ ] t-sys . This biphasic effect is not observed in presence of a ryanodine receptor (RyR) inhibitor, suggesting that above the hormetic zone, BTP2 impairs RyR function. Additionally, we found that BTP2 impairs the cytoplasmic Ca 2+ transients during repetitive excitation-contraction coupling (EC coupling) cycles independent of extracellular Ca 2+ entry. We determined that the release of Ca 2+ through the RyR was inhibited by BTP2, strongly suggesting that the RyR was the point of inhibition during the cycles of EC coupling. Conclusion. Our results show that both Ca 2+ channels, the Orai1 and RyR, are negatively regulated by BTP2, shedding new light on previous work that applied BTP2 to block SOCE in muscle.