Subcellular calcium variations are involved in physiological and pathological mechanisms. Whereas elementary calcium release events (CREs) have been known for almost three decades in intact muscle cells isolated from vertebrates, they remained not characterized in invertebrates until recently. Dynamic confocal imaging was used on intact skeletal muscle cells isolated enzymatically from the adult honeybee legs to characterize spatio-temporal features of subcellular CREs. The frequency of these insect CREs, measured in x–y time lapse series, was higher than frequencies usually described in vertebrates. Spatial spread at half maximum was larger than in vertebrates and had a slightly ellipsoidal shape, two characteristics that may be related to ultrastructural features specific to invertebrate cells. In line-scan experiments, the histogram of CREs’ duration followed a bimodal distribution, supporting the existence of both sparks and embers. Unlike in vertebrates, embers and sparks had similar amplitudes, a difference that could be related to genomic differences and/or excitation–contraction coupling specificities in honeybee skeletal muscle fibers. Arthropods muscle cells show strong genomic, ultrastructural and physiological differences with vertebrates and a comparative analysis may help to better understanding the roles and regulations of CREs. From a toxicological point of view, such a comparison will lead to better anticipating the myotoxicity of new insecticides targeting ryanodine receptors. Recent studies described the effects of these insecticides on macroscopic calcium homeostasis in bee neurons and muscle cells. Here, cyantraniliprole, the most recently approved anthranilic diamide in Europe, triggers calcium transients in bee muscle cell as well. Cyantraniliprole effects on Ca²⁺ sparks are currently under study.

Bilaterian voltage-gated Na⁺ channels (Na_V) evolved from voltage-gated Ca²⁺ channels (Ca_V). The Drosophila melanogaster Na⁺ channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between Ca_V and Na_V channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca²⁺-permeable Na⁺ channel belonging to the Na_V2 family because of its sequence similarity with Na_V channels. This is despite insect Na_V2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca²⁺ than Na⁺. In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd²⁺ and Zn²⁺. These characteristics are reminiscent of Ca_V channels. We also show a strong selectivity for Ca²⁺ and Ba²⁺ ions, marginal permeability to Li⁺, and impermeability to Mg²⁺ and Na⁺ ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as Ca_V4 rather than Na_V2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee’s channel, namely slow activation and inactivation kinetics and strong selectivity for Ca²⁺ ions.

The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca²+]_i) by inhibiting the release of calcium from the SR. The suppression of [Ca²+]_i was dose-dependent, with no effect at or below 1 μM and a 53 ± 8% (mean ± SEM, n = 9, cut fibers) attenuation at 0 mV with 25 μM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 ± 8%) and of the early peak component (46 ± 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I_Ca) were left, essentially, unaltered. However, the inactivation of I_Ca was slowed fourfold, and the conductance was reduced from 200 ± 16 to 143 ± 8 SF⁻¹ (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 ± 10% (n = 3) at 12 μM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30–50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.