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

Abstract

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.

Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy

“…In this regards, the use of SOCE inhibitors at different times of myogenesis process would be useful to explore the observed differences in resting Ca 2+ level between patient-derived muscle cells and control cells. Notably, in view of the non-specific effects mediated by SOCE inhibitors actually available (Le Guilcher et al, 2020;Meizoso-Huesca and Launikonis, 2021), focused studies will be required based on the use of several tools to unequivocally solve this issue.…”

Store-Operated Calcium Entry in Skeletal Muscle: What Makes It Different?

“…Despite its low selectivity, it is widely used as a SOCE inhibitor in skeletal muscle [ 38 , 41 , 87 , 110 , 190 , 191 ] at a concentration of 10 µM. Recently, a more detailed characterization of its effect in skeletal muscle fiber Ca 2+ handling was provided [ 192 ]. The authors observed that BTP2 affects Ca 2+ handling in rat skinned EDL fibers at multiple levels.…”

“…Thus, it blocked AP-induced SR Ca 2+ release and RyR leak at >10 µM. The impairment of RyR1 occurred in an indirect manner, probably by affecting the DHPR [ 192 ], which is a well-known target of other SOCE modulators (e.g., 2-APB and SKF96365 [ 168 ]).…”

“…), greatly increase the probability for off-target effects; (iii) the RyR plays a pivotal role in SOCE activation [30,39,59,89,97] but is characterized by substantial drug binding promiscuity toward small molecules and complex drug-receptor interactions, as exemplified by some of the better-studied molecules, namely, ryanodine and tetracaine (rev in [203]). In particular, the situation becomes delicate when considering fully mature muscle fibers, when all of the above applies, and interpretations solely based on drug action are not straightforward, as demonstrated, e.g., by Meizoso-Huesca [192]. Regarding pSOCE, another level of complexity is added when the whole EC-coupling machinery may constitute a potential drug target, including Ca v 1.1 and Na v 1.4, which are known to be affected by current SOCE pharmacology.…”

Alteration of STIM1/Orai1-Mediated SOCE in Skeletal Muscle: Impact in Genetic Muscle Diseases and Beyond

“…Recently, a variety of new small molecules blocking CRAC channels have been identified and developed, such as pyrtriazoles or pyrazole SKF-96365 analogues [131,170]. However, all currently available SOCE inhibitors show no specific effects [171,172].…”