In intact muscle fibers, functional properties of ryanodine receptor (RYR)–mediated sarcoplasmic reticulum (SR) Ca2+ release triggered by activation of the voltage sensor CaV1.1 have so far essentially been addressed with diffusible Ca2+-sensitive dyes. Here, we used a domain (T306) of the protein triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane, in the immediate vicinity of RYR channels, within the triad region. Fluorescence of untargeted GCaMP6f was distributed throughout the muscle fibers and experienced large Ca2+-dependent changes, with obvious kinetic delays, upon application of voltage-clamp depolarizing pulses. Conversely, T306-GCaMP6f localized to the triad and generated Ca2+-dependent fluorescence transients of lower amplitude and faster kinetics for low and intermediate levels of Ca2+ release than those of untargeted GCaMP6f. By contrast, model simulation of the spatial gradients of Ca2+ following Ca2+ release predicted limited kinetic differences under the assumptions that the two probes were present at the same concentration and suffered from identical kinetic limitations. At the spatial level, T306-GCaMP6f transients within distinct regions of a same fiber yielded a uniform time course, even at low levels of Ca2+ release activation. Similar observations were made using GCaMP6f fused to the γ1 auxiliary subunit of CaV1.1. Despite the probe's limitations, our results point out the remarkable synchronicity of voltage-dependent Ca2+ release activation and termination among individual triads and highlight the potential of the approach to visualize activation or closure of single groups of RYR channels. We anticipate targeting of improved Ca2+ sensors to the triad will provide illuminating insights into physiological normal RYR function and its dysfunction under stress or pathological conditions.
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5 April 2021
Article|
February 04 2021
Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad
Colline Sanchez
,
Colline Sanchez
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
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Christine Berthier
,
Christine Berthier
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
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Yves Tourneur
,
Yves Tourneur
2
Departamento Nutrição, Universidade Federal de Pernambuco, Recife, Brazil
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Laloé Monteiro
,
Laloé Monteiro
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
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Bruno Allard
,
Bruno Allard
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
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Laszlo Csernoch
,
Laszlo Csernoch
3
Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
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Vincent Jacquemond
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
Correspondence to Vincent Jacquemond: vincent.jacquemond@univ-lyon1.fr
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Colline Sanchez
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
Christine Berthier
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
Yves Tourneur
2
Departamento Nutrição, Universidade Federal de Pernambuco, Recife, Brazil
Laloé Monteiro
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
Bruno Allard
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
Laszlo Csernoch
3
Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
Vincent Jacquemond
1
Université Lyon, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique UMR-5310, Institut National de la Santé et de la Recherche Médicale U-1217, Institut NeuroMyoGène, Lyon, France
Correspondence to Vincent Jacquemond: vincent.jacquemond@univ-lyon1.fr
Received:
February 14 2020
Revision Received:
December 03 2020
Accepted:
December 23 2020
Online Issn: 1540-7748
Print Issn: 0022-1295
Funding:
AFM-Téléthon
(2.3.1.3)
Centre National de la Recherche Scientifique
(NO AWARD)
Institut National de la Santé et de la Recherche Médicale
(NO AWARD)
Université Claude Bernard Lyon 1
(NO AWARD)
© 2021 Sanchez et al.
2021
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 (4): e202012592.
Article history
Received:
February 14 2020
Revision Received:
December 03 2020
Accepted:
December 23 2020
Citation
Colline Sanchez, Christine Berthier, Yves Tourneur, Laloé Monteiro, Bruno Allard, Laszlo Csernoch, Vincent Jacquemond; Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad. J Gen Physiol 5 April 2021; 153 (4): e202012592. doi: https://doi.org/10.1085/jgp.202012592
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