The Orai1 inhibitor BTP2 has multiple effects on Ca²⁺ handling in skeletal muscle

BTP2 is an inhibitor of the Ca²⁺ channel Orai1, which mediates store-operated Ca²⁺ entry (SOCE). Despite having been extensively used in skeletal muscle, the effects of this inhibitor on Ca²⁺ handling in muscle cells have not been described. To address this question, we used intra- and extracellular application of BTP2 in mechanically skinned fibers and developed a localized modulator application approach, which provided in-preparation reference and test fiber sections to enhance detection of the effect of Ca²⁺ handling modulators. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of resting extracellular Ca²⁺ flux. Increasing concentrations of BTP2 caused a shift from inducing accumulation of Ca²⁺ in the t-system due to Orai1 blocking to reducing the resting [Ca²⁺] in the sealed t-system. This effect was not observed in the absence of functional ryanodine receptors (RYRs), suggesting that higher concentrations of BTP2 impair RYR function. Additionally, we found that BTP2 impaired action potential–induced Ca²⁺ release from the sarcoplasmic reticulum during repetitive stimulation without compromising the fiber Ca²⁺ content. BTP2 was found to have an effect on RYR-mediated Ca²⁺ release, suggesting that RYR is the point of BTP2-induced inhibition during cycles of EC coupling. The effects of BTP2 on the RYR Ca²⁺ leak and release were abolished by pre-exposure to saponin, indicating that the effects of BTP2 on the RYR are not direct and require a functional t-system. Our results demonstrate the presence of a SOCE channels–mediated basal Ca²⁺ influx in healthy muscle fibers and indicate that BTP2 has multiple effects on Ca²⁺ handling, including indirect effects on the activity of the RYR.