Dantrolene Requires Mg2+ and ATP To Inhibit the Ryanodine Receptor.

Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca2+ release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca2+ release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca2+ release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca2+ release experiments demonstrated that the effect of dantrolene and Mg2+ was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 µM dantrolene reduced RyR channel open probability by ∼50% in the presence of 3 mM free Mg2+ and 1 mM ATP, whereas channel activity further decreased to ∼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg2+-dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR.

Expression of BK channels and Na+-K+ pumps in the apical membrane of lacrimal acinar cells suggests a new molecular mechanism for primary tear-secretion.

PURPOSE: Primary fluid secretion in secretory epithelia relies on the unidirectional transport of ions and water across a single cell layer. This mechanism requires the asymmetric apico-basal distribution of ion transporters and intracellular Ca2+ signaling. The primary aim of the present study was to verify the localization and the identity of Ca2+-dependent ion channels in acinar cells of the mouse lacrimal gland. METHODS: Whole-cell patch-clamp-electrophysiology, spatially localized flash-photolysis of Ca2+ and temporally resolved digital Ca2+-imaging was combined. Immunostaining of enzymatically isolated mouse lacrimal acinar cells was performed. RESULTS: We show that the Ca2+-dependent K+-conductance is paxilline-sensitive, abundant in the luminal, but negligible in the basal membrane; and co-localizes with Cl--conductance. These data suggest that both Cl- and K+ are secreted into the lumen and thus they account for the high luminal [Cl-] (∼141 mM), but not for the relatively low [K+] (<17 mM) of the primary fluid. Accordingly, these results also imply that K+ must be reabsorbed from the primary tear fluid by the acinar cells. We hypothesized that apically-localized Na+-K+ pumps are responsible for K+-reabsorption. To test this possibility, immunostaining of lacrimal acinar cells was performed using anti-Na+-K+ ATP-ase antibody. We found positive fluorescence signal not only in the basal, but in the apical membrane of acinar cells too. CONCLUSIONS: Based on these results we propose a new primary fluid-secretion model in the lacrimal gland, in which the paracellular pathway of Na+ secretion is supplemented by a transcellular pathway driven by apical Na+-K+ pumps.