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.
Departamento Nutrição, Universidade Federal de Pernambuco, Recife, Brazil.
J Gen Physiol. 2021 Apr 5;153(4). doi: 10.1085/jgp.202012592.
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.
在完整的肌纤维中，钙通道电压传感器 CaV1.1 激活触发的兰尼碱受体（RYR）介导的肌质网（SR）Ca2+释放的功能特性，迄今为止主要是通过扩散性 Ca2+敏感染料来研究的。在这里，我们使用蛋白三联蛋白（triadin）的一个结构域（T306）将 Ca2+敏感探针 GCaMP6f 靶向到连接 SR 膜，即 RYR 通道附近的三联区。未靶向的 GCaMP6f 的荧光分布在整个肌纤维中，并在应用电压钳去极化脉冲时经历大的 Ca2+依赖性变化，具有明显的动力学延迟。相反，T306-GCaMP6f 定位于三联区，并产生 Ca2+依赖性荧光瞬变，其幅度低于未靶向 GCaMP6f，动力学更快，在低水平和中等水平的 Ca2+释放时比未靶向 GCaMP6f 更快。相比之下，根据两个探针处于相同浓度且具有相同动力学限制的假设，对 Ca2+释放后 Ca2+空间梯度的模型模拟预测动力学差异有限。在空间水平上，同一纤维的不同区域内的 T306-GCaMP6f 瞬变产生均匀的时程，即使在低水平的 Ca2+释放激活时也是如此。使用融合到 CaV1.1 的 γ1 辅助亚基的 GCaMP6f 也观察到了类似的结果。尽管该探针存在局限性，但我们的结果指出了单个三联体之间电压依赖性 Ca2+释放激活和终止的显著同步性，并强调了该方法可视化单个 RYR 通道组的激活或关闭的潜力。我们预计将改进的 Ca2+传感器靶向三联区将为生理正常 RYR 功能及其在应激或病理条件下的功能障碍提供有启发性的见解。
