Evidence for Status Epilepticus and Pro-Inflammatory Changes after Intranasal Kainic Acid Administration in Mice.

Kainic acid (KA) is routinely used to elicit status epilepticus (SE) and epileptogenesis. Among the available KA administration protocols, intranasal instillation (IN) remains understudied. Dosages of KA were instilled IN in mice. Racine Scale and Video-EEG were used to assess and quantify SE onset. Time spent in SE and spike activity was quantified for each animal and confirmed by power spectrum analysis. Immunohistochemistry and qPCR were performed to define brain inflammation occurring after SE, including activated microglial phenotypes. Long term video-EEG recording was also performed. Titration of IN KA showed that a dose of 30 mg/kg was associated with low mortality while eliciting SE. IN KA provoked at least one behavioral and electrographic SE in the majority of the mice (>90%). Behavioral and EEG SE were accompanied by a rapid and persistent microglial-astrocytic cell activation and hippocampal neurodegeneration. Specifically, microglial modifications involved both pro- (M1) and anti-inflammatory (M2) genes. Our initial long-term video-EEG exploration conducted using a small cohort of mice indicated the appearance of spike activity or SE. Our study demonstrated that induction of SE is attainable using IN KA in mice. Typical pro-inflammatory brain changes were observed in this model after SE, supporting disease pathophysiology. Our results are in favor of the further development of IN KA as a means to study seizure disorders. A possibility for tailoring this model to drug testing or to study mechanisms of disease is offered.

Pharmacological characterization and molecular determinants of the activation of transient receptor potential V2 channel orthologs by 2-aminoethoxydiphenyl borate.

Despite its expression in different cell types, transient receptor potential V2 (TRPV2) is still the most cryptic members of the TRPV channel family. 2-Aminoethoxydiphenyl borate (2APB) has been shown to be a common activator of TRPV1, TRPV2, and TRPV3, but 2APB-triggered TRPV2 activation remains to be thoroughly characterized. In this study, we have developed an assay based on cell lines stably expressing mouse TRPV2 channels and intracellular calcium measurements to perform a pharmacological profiling of the channel. Phenyl borate derivatives were found to activate mouse TRPV2 with similar potencies and thus were used to screen a panel of channel blockers. Besides the classic TRP inhibitors ruthenium red (RR) and 1-(beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF96365), two potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine, and an inhibitor of capacitative calcium entry, 1-(2-(trifluoromethyl) phenyl) imidazole (TRIM), were found to inhibit TRPV2 activation by 100 microM 2APB. Activation by 300 microM 2APB, however, could only be inhibited by RR and TRIM. Electrophysiological recordings demonstrated that TEA inhibition was use-dependent, suggesting that high concentrations of 2APB might induce a progressive conformational change of the channel. Comparison of TRPV2 orthologs revealed that the human channel was insensitive to 2APB. Analysis of chimeric constructs of mouse and human TRPV2 channels showed that the molecular determinants of 2APB sensitivity could be localized to the intracellular amino and carboxyl domains. Finally, using lentiviral-driven expression, we demonstrate that hTRPV2 exerts a dominant-negative effect on 2APB activation of native rodent TRPV2 channels and thus may provide an interesting tool to investigate cellular functions of TRPV2 channels.

PI3-kinase promotes TRPV2 activity independently of channel translocation to the plasma membrane.

Cellular or chemical activators for most transient receptor potential channels of the vanilloid subfamily (TRPV) have been identified in recent years. A remarkable exception to this is TRPV2, for which cellular events leading to channel activation are still a matter of debate. Diverse stimuli such as extreme heat or phosphatidylinositol-3 kinase (PI3-kinase) regulated membrane insertion have been shown to promote TRPV2 channel activity. However, some of these results have proved difficult to reproduce and may underlie different gating mechanisms depending on the cell type in which TRPV2 channels are expressed. Here, we show that expression of recombinant TRPV2 can induce cytotoxicity that is directly related to channel activity since it can be prevented by introducing a charge substitution in the pore-forming domain of the channel, or by reducing extracellular calcium. In stably transfected cells, TRPV2 expression results in an outwardly rectifying current that can be recorded at all potentials, and in an increase of resting intracellular calcium concentration that can be partly prevented by serum starvation. Using cytotoxicity as a read-out of channel activity and direct measurements of cell surface expression of TRPV2, we show that inhibition of the PI3-kinase decreases TRPV2 channel activity but does not affect the trafficking of the channel to the plasma membrane. It is concluded that PI3-kinase induces or modulates the activity of recombinant TRPV2 channels; in contrast to the previously proposed mechanism, activation of TRPV2 channels by PI3-kinase is not due to channel translocation to the plasma membrane.

[The TRP family of channels: a complex gallery of characters]. / La famille des canaux TRP: une galerie complexe de portraits.

Calcium influxes are of fundamental importance in eukaryotic cell functions. These calcium influxes are carried by different classes of membrane proteins that allow regulated calcium entry. If in excitable cells, such as neurones or muscle, voltage-dependent calcium channels represent the main source of calcium influx, other proteins are needed to assume such a function in non-excitable cells. In these, a sustained calcium influx is observed, secondary to phospholipase C activation, IP3 synthesis and internal calcium release. The identity of proteins implicated in this second messenger calcium-driven influx, as well as the mechanisms of activation of these channels have long been debated. In recent years, genes encoding a new kind of cationic channels called TRP channels have been identified. This molecular work has set the basis for further functional studies and helped to gain crucial information on the mechanisms by which extracellular calcium can penetrate into non-excitable cells. This review will present the most recent advances obtained on the molecular diversity of TRP channels and their mode of gating.