Role of FK506-binding protein in Ca^(2+) spark regulation

The elementary Ca^(2+) release events, Ca^(2+) sparks, has been found for a quarter of century. However, the molecular regulation of the spark generator, the ryanodine receptor(RyR) on the sarcoplasmic reticulum,remains obscure. Although each subunit of the RyR homotetramer has a site for FK506-binding protein(FKBP), the role of FKBPs in modifying RyR Ca^(2+) sparks has been debated for long. One of the reasons behind the controversy is that most previous studies detect spontaneous sparks, where the mixture with out-of-focus events and local wavelets prevents an accurate characterization of Ca^(2+) sparks. In the present study, we detected Ca^(2+) sparks triggered by single L-type Ca^(2+) channels(LCCs) under loose-seal patch clamp conditions in FK506-treated or FKBP12.6 knockout cardiomyocytes. We found that FKBP dissociation both by FK506 and by rapamycin decreased the Ca^(2+) spark amplitude in ventricular cardiomyocytes. This change was neither due to decreased releasable Ca^(2+) in the sarcoplasmic reticulum,nor explained by changed RyR sensitivity. Actually FK506 increased the LCC-RyR coupling probability and curtailed the latency for an LCC to trigger a RyR Ca^(2+) spark. FKBP12.6 knockout had similar effects as FK506/rapamycin treatment, indicating that the decreased spark amplitude was attributable to the dissociation of FKBP12.6 rather than FKBP12. We also explained how decreased amplitude of spontaneous sparks after FKBP dissociation sometimes appears to be increased or unchanged due to inappropriate data processing. Our results provided firm evidence that without the inter-RyR coordination by functional FKBP12.6, the RyR recruitment during a Ca^(2+) spark would be compromised despite the sensitization of individual RyRs.

The formation of Ca^(2+) gradients at the cleavage furrows during cytokinesis of Zebrafish embryos

In dividing embryos,a localized elevation in intracellular Ca^(2+)([Ca^(2+)]i)at the cleavage furrow has been shown to be essential for cytokinesis.However,the underlying mechanisms for generating and maintaining these[Ca^(2+)]_(i) gradients throughout cytokinesis are not fully understood.In the present study,we analyzed the role of inositol 1,4,5-trisphosphate receptors(IP3Rs)and endoplasmic reticulum(ER)distribution in determining the intracellular Ca^(2+) gradients in early zebrafish blastomeres.Application of the injected Ca^(2+) indicator,Indo-1,showed that during the first cell division a standing Ca^(2+) gradient was formed~35 min after fertilization,with the[Ca^(2+)]_(i) spatially decaying from 500–600 nmol/L at the cleavage furrow to 100–200 nmol/L around the nucleus.While the IP3R immunohistochemical fluorescence was relatively concentrated in the peri-furrow region,ER labeling was relatively enriched in both peri-furrow and peri-nuclear regions.Numeric simulation suggested that a divergence in the spatial distribution of IP3R and the locations of Ca^(2+) uptake within the ER was essential for the formation of a standing Ca^(2+) gradient,and the Ca^(2+) gradient could only be well-established under an optimal stoichiometry of Ca^(2+) uptake and release.Indeed,while inhibition of IP3R Ca^(2+) release blocked the generation of the Ca^(2+)gradient at a lower[Ca^(2+)]_(i) level,both Ca^(2+) release stimulation by inositol 1,4,5-trisphosphate(IP3)injection and ER Ca^(2+) pump inhibition by cyclopiazonic acid also eliminated the Ca^(2+) gradients at higher[Ca^(2+)]_(i) levels.Our results suggest a dynamic relationship between ER-mediated Ca^(2+) release and uptake that underlies the maintenance of the perifurrow Ca^(2+) gradient and is essential for cytokinesis of zebrafish embryos.