Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons.

The lipid diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to verify the existence of DAG-sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca(2+) ([Ca(2+)]i) in nearly 35% of the KCl-responsive cells. These Ca(2+) responses disappeared in a Ca(2+)-free medium supplemented with EGTA. Mn(2+) quench experiments showed that OAG activated Ca(2+)-conducting channels that were also permeant to Ba(2+). The OAG-induced Ca(2+) responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[beta-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd(3+). Replacing Na(+) ions with N-methyl-D-glucamine diminished the amplitude of the OAG-induced Ca(2+) responses showing that the Ca(2+) entry was mediated via Na(+)-dependent and Na(+)-independent mechanisms. Experiments carried out with the fluorescent Na(+) indicator CoroNa Green showed that OAG elevated [Na(+)]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca(2+)]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca(2+) responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C-type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca(2+)]i. Whole-cell patch-clamp recordings showed that hyperforin activated non-selective cation channels. They were blocked by SKF-96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin- and OAG-sensitive Ca(2+)-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.

Serum and glucocorticoid inducible kinases in the regulation of the cardiac sodium channel SCN5A.

The serum and glucocorticoid inducible kinase SGK1 and its isoform SGK3 are both expressed in cardiac tissue. One of the functions of SGK1 is the phosphorylation and inactivation of the ubiquitin ligase Nedd4-2, which in turn could be shown to downregulate the voltage-gated Na+ channel SCN5A (hH1). The present study has been performed to test for a role of SGK1 and SGK3 in the regulation of SCN5A. To this end cRNA encoding the human Na+ channel SCN5A was injected into Xenopus laevis oocytes with or without cRNA encoding the wild-type kinases SGK1, the constitutively active kinase (S422D)SGK1, the inactive form K127NSGK1 or the wild-type SGK3. SCN5A currents were activated by coexpression of either wild-type SGK1 or SGK3 or the constitutively active S422DSGK1. In contrast, the inactive mutant K127NSGK1 significantly decreased the currents. Moreover, coexpression of SGK3 significantly altered SCN5A gating, i.e. it hyperpolarized the activation threshold and depolarized the prepotential required for 50% availability of the channel. Opposite shifts of gating properties were elicited by mutation of serine to alanine (S483ASCN5A and S663ASCN5A) in the SGK consensus sequences of SCN5A. The present observations disclose a role of the kinases SGK1 and SGK3 in the regulation of cardiac Na+ channels. As SGK1 is upregulated by glucocorticoids, mineralocorticoids and a variety of inflammatory mediators and both kinases are activated by insulin and IGF1, the kinases could mediate effects of those hormones and mediators on cardiac function.