Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation–contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca²⁺ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca²⁺ handling and EC coupling: acute application of probenecid generates a rise in resting Ca²⁺ that also occurs in nominally Ca²⁺-free extracellular medium. This effect is associated with a reduction of Ca²⁺ release through the sarcoplasmic reticulum (SR) Ca²⁺ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca²⁺ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca²⁺ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.

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.