The Biological Role of the ζ Subunit as Unidirectional Inhibitor of the F1FO-ATPase of Paracoccus denitrificans.

The biological roles of the three natural F1FO-ATPase inhibitors, ε, ζ, and IF1, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF1, but in the F1FO-ATPase of Paracoccus denitrificans (PdF1FO), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ) with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdF1FO-ATPase activity without affecting the PdF1FO-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdF1FO-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.

STIM1 and Orai1 mediate thrombin-induced Ca(2+) influx in rat cortical astrocytes.

In astrocytes, thrombin leads to cytoplasmic Ca(2+) elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca(2+) increase generated by an interplay between ER-Ca(2+) release and store-operated Ca(2+) entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca(2+) depletion and binds Orai1 to activate Ca(2+) influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca(2+) response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca(2+) influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca(2+) influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca(2+) response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca(2+) release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca(2+) influx.

Depolarization, exocytosis and amino acid release evoked by hyposmolarity from cortical synaptosomes.

External osmolarity reduction (20%) led to labelled glutamate, GABA and taurine release from rat brain cortical synaptosomes. A Cl--independent, Na+-dependent, La3+-sensitive and tetrodotoxin (TTX) reduced depolarization of synaptosomes occurred upon hyposmolarity, suggestive of Na+ entry through nonselective cation channels. This depolarization, together with cytosolic Ca2+ ([Ca2+]I) increase, resulted in exocytosis, monitored by FM1-43. The release fraction resulting from these phenomena was estimated, by its decrease, by La3+, EGTA-AM and tetanus toxin (TeTX), as 34-44% for glutamate, 21-29% for GABA and 18-22% for taurine. Protein kinase C (PKC) activation by phorbol-12-myristate-13-acetate (PMA) increased the hyposmolarity-elicited exocytosis and this activation increased glutamate (80%), GABA (51%) and taurine (42%) hyposmotic efflux. Inhibition by chelerythrine reduced glutamate, GABA and taurine efflux by 64%, 50% and 24%, respectively. The Na+-dependence of amino acid release (glutamate 63%, GABA 46% and taurine 29%) may result from both, prevention of the depolarization-exocytosis efflux, and blockade of the carrier reversal operation. Carrier blockade by dl-threo-beta-benzyloxy aspartate (TBOA) and NO-711 resulted in 37% and 28% reduction of glutamate and GABA release, respectively. Contribution of the osmolyte leak pathway to amino acid release, estimated by the influence of Cl- (NPPB) and tyrosine kinase (AG18) blocker, was up to 55% for taurine, but only 10-18% for GABA, with apparently no contribution for glutamate. The predominant osmolyte-type mechanism of taurine release suggest its function in volume control in nerve endings, while glutamate and GABA respond to events concurrent with hyposmolarity by a neurotransmitter-like release mechanism. The hyposmolarity-induced amino acid efflux from nerve endings may have consequences for neuronal excitability during hyponatremia.

Osmolytes and mechanisms involved in regulatory volume decrease under conditions of sudden or gradual osmolarity decrease.

A decrease in external osmolarity results in cell swelling and the immediate activation of a mechanism to restore cell volume, known as regulatory volume decrease (RVD). When exposed to a gradual osmolarity decrease (GODE), some cells do not swell. This reflects the operation of an active regulatory process known as isovolumetric regulation (IVR). The mechanisms underlying IVR appear similar to those activated during RVD, namely the extrusion of K+, Cl-, amino acids, and other organic molecules. A previous study has documented IVR in cerebellar granule neurons, parallel to an early efflux of taurine and Cl-, whereas K+ efflux is delayed. In this work we briefly review the importance of amino acids in the mechanisms of cell volume control in the brain, with emphasis on IVR. We also present experiments showing the response to GODE in cerebellar astrocytes. The currents activated during GODE, recorded in the whole-cell configuration of the patch clamp technique, indicate the early activation of an anion current, followed by a more delayed cation current. A correlation between the time course of amino acid efflux during GODE and the occurrence or not of IVR in various cell types, suggest the importance of these osmolytes in the volume regulatory process in this model.