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.
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4 January 2021
Article|
December 14 2020
The Orai1 inhibitor BTP2 has multiple effects on Ca2+ handling in skeletal muscle
Aldo Meizoso-Huesca,
Aldo Meizoso-Huesca
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
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Bradley S. Launikonis
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
Correspondence to Bradley S. Launikonis: b.launikonis@uq.edu.au
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Aldo Meizoso-Huesca
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
Bradley S. Launikonis
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
Correspondence to Bradley S. Launikonis: b.launikonis@uq.edu.au
Received:
August 23 2020
Revision Received:
October 21 2020
Accepted:
November 17 2020
Online Issn: 1540-7748
Print Issn: 0022-1295
Funding:
Australian Research Council
(DP180100937)
© 2020 Meizoso-Huesca and Launikonis
2020
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
J Gen Physiol (2021) 153 (1): e202012747.
Article history
Received:
August 23 2020
Revision Received:
October 21 2020
Accepted:
November 17 2020
Citation
Aldo Meizoso-Huesca, Bradley S. Launikonis; The Orai1 inhibitor BTP2 has multiple effects on Ca2+ handling in skeletal muscle. J Gen Physiol 4 January 2021; 153 (1): e202012747. doi: https://doi.org/10.1085/jgp.202012747
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