No abstract

No abstract

The Golgi apparatus (GA) is a dynamic intracellular Ca(2+) store endowed with complex Ca(2+) homeostatic mechanisms in part distinct from those of the endoplasmic reticulum (ER). We describe the generation of a novel fluorescent Ca(2+) probe selectively targeted to the medial-Golgi. We demonstrate that in the medial-Golgi: (i) Ca(2+) accumulation takes advantage of two distinct pumps, the sarco/endoplasmic reticulum Ca(2+) ATPase and the secretory pathway Ca(2+) ATPase1; (ii) activation of IP3 or ryanodine receptors causes Ca(2+) release, while no functional two-pore channel was found; (iii) luminal Ca(2+) concentration appears higher than that of the trans-Golgi, but lower than that of the ER, suggesting the existence of a cis- to trans-Golgi Ca(2+) concentration gradient. Thus, the GA represents a Ca(2+) store of high complexity where, despite the continuous flow of membranes and luminal contents, each sub-compartment maintains its Ca(2+) identity with specific Ca(2+) homeostatic characteristics. The functional role of such micro-heterogeneity in GA Ca(2+) handling is discussed.

No abstract

Mice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca2+ leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca2+ uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca2+ concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca2+ release from SR during repetitive electrical stimulation or caffeine administration. Also mitochondrial ROS production associated with contractile activity (detected with Mitosox probe) was much higher in YS fibers than in WT. Importantly, the inhibition of mitochondrial Ca2+ uptake achieved by silencing MCU reduced ROS accumulation in the matrix and Ca2+ release from SR. Finally, inhibition of mitochondrial ROS accumulation using Mitotempo reduced SR Ca2+ release in YS fibers exposed to caffeine. The present results support the view that mitochondria take up larger amounts of Ca2+ in YS than in WT fibers and that mitochondrial ROS production substantially contributes to the increased caffeine-sensitivity and to the enhanced Ca2+ release from SR in YS fibers.