Francisco Javier Martin-Romero

STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca(2+) concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although the molecular details for STIM1-SOC binding is known, the regulation of SOCE remains largely unknown. A detailed list of phosphorylated residues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phosphospecific antibodies, we demonstrate that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608 and
Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type
STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.

From 1 to 3 h after the onset of cerebellar granule cells (CGC) apoptosis in a low-K+(5 mm KCl) medium there was a large decay of NADH and a 2.5-fold increase of the rate of reactive oxygen species (ROS) production (measured using CGC loaded with dichlorodihydrofluorescein). During the same time period, the ascorbate-dependent NADH oxidase activity, which accounted for more than 90% of both total NADH oxidase activity and NADH-dependent *O2- production of CGC lysates, increased 2.5- to threefold. The stimulation of the ascorbate-dependent NADH oxidase activity by oxidized cytochrome c, 2.5-fold at saturation with a K(0.5) of 4-5 microm cytochrome c, can at least partially explain this activation. The plasma membrane ascorbate-dependent NADH oxidase activity accounted for more than 70% of the total activity (both in terms of NADH oxidase and *O2- release) of CGC lysates. 4-Hydroxyquinazoline (4-HQ), which was found to block this apoptotic process, prevented the increase of ROS production. 4-HQ protection against cell viability loss and DNA fragmentation correlated with the inhibition by 4-HQ of the ascorbate-dependent NADH oxidase activity of CGC lysates, showing the same K(0.5)-value (4-5 mm 4-HQ). The efficient blockade of CGC apoptosis by addition of superoxide dismutase to the medium further supports the neurotoxic role of *O2- overproduction by the plasma membrane ascorbate-dependent NADH oxidase.

Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37 degrees C with 10 microM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg2+ concentration ([Mg2+]i) was found to be dependent on external Mg2+ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg2+ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na+ gradient across the plasma membrane of synaptosomes by treatment with the Na+ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg2+]i, from 1.0 to 1.2-1.3 mM in an incubation medium containing 5 mM Mg2+. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg2+]i. Reversal of the Na+ gradient by incubation of synaptosomes in a medium in which external Na+ was replaced by choline increased [Mg2+]i up to 1.6 and 2.2 mM for extrasynaptosomal Mg2+ concentrations of 1 and 8 mM, respectively. We conclude that a Na+/Mg2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg2+]i from 1.1 +/- 0.1 up to 1.6 +/- 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5'-(beta gamma-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg2+]i. It is concluded that Mg2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.