Detection of Ca²⁺ transients near ryanodine receptors by targeting fluorescent Ca²⁺ sensors to the triad

In intact muscle fibers, functional properties of ryanodine receptor (RYR)–mediated sarcoplasmic reticulum (SR) Ca²⁺ release triggered by activation of the voltage sensor Ca_V1.1 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 Ca_V1.1. 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.