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- Materials and methods
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 Ca2+ ([Ca2+]i) in nearly 35% of the KCl-responsive cells. These Ca2+ responses disappeared in a Ca2+-free medium supplemented with EGTA. Mn2+ quench experiments showed that OAG activated Ca2+-conducting channels that were also permeant to Ba2+. The OAG-induced Ca2+ responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[β-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd3+. Replacing Na+ ions with N-methyl-d-glucamine diminished the amplitude of the OAG-induced Ca2+ responses showing that the Ca2+ 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 [Ca2+]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca2+ 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 [Ca2+]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 Ca2+-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.
The lipid diacylglycerol (DAG) is a second messenger involved in key cellular processes [for a review see (Carrasco and Merida 2007)]. Several DAG-sensitive proteins have been identified like some isoforms of the protein kinase C (PKC) family, Munc13 proteins (Brose et al. 2004) and ion channels of the C-type transient receptor potential protein family (TRPC). Seven TRPC are now identified and named TRPC1 to TRPC7. They all function as cation channels but they exhibit distinct gating and biophysical properties. For instance, homomeric TRPC1, TRPC3, TRPC6 or TRPC7 channels and heteromeric TRPC1–TRPC3 and TRPC3–TRPC4 channels can open in response to DAG application allowing an entry of Ca2+ into the cell (Hofmann et al. 1999; Lintschinger et al. 2000; Liu et al. 2005; Poteser et al. 2006). This DAG-dependent activation of TRPC channels occurs in a PKC-independent manner.
Several studies reported the existence of DAG-sensitive channels in neural cells. For instance, DAG activates Ca2+-conducting channels in the neuronal cell lines PC12 (Mwanjewe and Grover 2004) and IMR-32 (Nasman et al. 2006), as well as in cortical astrocytes (Grimaldi et al. 2003; Beskina et al. 2007), vomeronasal neurons (Lucas et al. 2003), hippocampal neurons (Tai et al. 2008) and neural stem cells (Pla et al. 2005). In astrocytes from embryonic rat brains, TRPC3 channels mediate the DAG-induced cytosolic Ca2+ changes (Grimaldi et al. 2003) whereas in astrocytes prepared from embryonic murine brains TRPC6 forms the DAG-sensitive channels (Beskina et al. 2007). On the other hand, the Ca2+ responses are due to TRPC2 in vomeronasal neurons (Lucas et al. 2003) and to TRPC6 in the hippocampus (Tai et al. 2008). These latter findings suggest that the molecular identity of the DAG-sensitive channels seems species- and tissue-dependent. At E13, the immature cortex expresses all TRPC isoforms (Boisseau, Kunert-Keil, Lucke and Bouron; unpublished data). We thus tried to determine whether the first cortical neurons, which are generated at E11–12 (Kriegstein and Noctor 2004), express functional DAG-sensitive channels. By recording cytosolic Ca2+ changes we observed that the DAG analogues 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) caused a Ca2+ influx via channels sensitive to Gd3+ and SKF-96365 but insensitive to nifedipine and omega-conotoxin GVIA (ω-CTx), (Sigma-Aldrich). The OAG-induced Ca2+ responses were observed in KCl-responding and KCl-insensitive cells. Similarly to OAG or SAG, the DAG lipase inhibitor RHC80267, used to prevent the degradation of DAG, elevated [Ca2+]i but not the PKC activator phorbol 12-myristate 13-acetate (PMA). Moreover, the OAG-induced responses were not altered by PKC activation or inhibition. This shows that OAG recruited SKF-96365-sensitive Ca2+-conducting channels in a PKC-independent manner. Flufenamic acid (FFA) was used to further characterize the identity of the OAG-sensitive channels. FFA increases the amplitude of currents through TRPC6 channels but blocks TRPC3 and TRPC7 channels (Inoue et al. 2001). In cultured cortical neurons, FFA potentiated the OAG-induced Ca2+ responses suggesting that TRPC6 are key constituents of the OAG-sensitive channels. In situ hybridization experiments confirmed the presence of TRPC6, distributed throughout the cortical wall (preplate and ventricular zone) including in cortical neurons. Furthermore, Ca2+ imaging experiments and whole-cell patch-clamp recordings showed that hyperforin, a specific activator of TRPC6 channels (Leuner et al. 2007), activated non-selective cation channels blocked by SKF-96365. Currents through hyperforin-activated channels were increased by FFA. Altogether, these data suggest that OAG activated TRPC6 channels or channels exhibiting TRPC6-like properties. This is the first description of functional second messenger-operated channels in cortical neurons.
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- Materials and methods
By performing Ca2+ imaging and electrophysiological experiments we have shown that in cortical cells from E13 mouse embryos OAG (or SAG) and hyperforin activate plasma membrane cation channels. These OAG-induced Ca2+ responses were observed in KCl-responsive and in KCl-unresponsive cells. This latter cell population, considered as non-neuronal cells, was not further analysed as recent studies already demonstrated that OAG triggers an entry of Ca2+ in cortical astrocytes. It develops via TRPC3 channels in cortical astrocytes prepared from E17 rat embryos (Grimaldi et al. 2003) and via TRPC6 channels in cortical astrocytes prepared from E17 mouse embryos (Beskina et al. 2007). In addition, an OAG-induced Ca2+ entry is found in neural stem cells prepared from E13 rat embryos (Pla et al. 2005). Thus, our data, showing a OAG-induced Ca2+ entry in KCl-unresponsive cells (e.g. cell 2 in Fig. 1) is in line with reports describing OAG-induced Ca2+ signals in non-neuronal cortical cells (Grimaldi et al. 2003; Beskina et al. 2007). Therefore, we focused our analysis on the KCl-responsive cells. To our knowledge, this is the first report showing second messenger-operated channels in cortical neurons.
Nearly 35% of the KCl-responsive cells responded to OAG. In most cases, OAG induced Ca2+ oscillations even in cells treated with caffeine to deplete the caffeine-sensitive Ca2+ pool of the endoplasmic reticulum (not shown). The OAG-induced Ca2+ signals, seen in freshly dissociated cells as well as in cultured isolated cells kept up to six DIV, disappeared when Ca2+ was omitted from the extracellular milieu. The entry of Ca2+ could be triggered by OAG, SAG or in the presence of the DAG lipase inhibitor RHC80267. This Ca2+ route was unaffected by the voltage-gated Ca2+ channel antagonists nifedipine and ω-CTx but it was strongly blocked by Gd3+ and SKF-96365. Replacing Na+ ions with NMDG+ did not suppress but attenuated the OAG-induced Ca2+ rise. Analysis of cytosolic Na+ changes with the fluorescent Na+ indicator CoroNa Green revealed that OAG caused an entry of Na+. A similar observation was made in vascular smooth muscle cells (Poburko et al. 2007). Based on these findings it is proposed that DAG controls the activity of SKF-96365-sensitive channels allowing a Ca2+ entry via Na+-dependent and Na+-independent mechanisms. As the cytosolic Ca2+ rise partially depends on the extracellular concentration of Na+, OAG controls the activity of Na+- and Ca2+-conducting channels. The intracellular load of Na+ thus activates the Na+/Ca2+ exchanger which, in turn, permits an entry of Ca2+ (Platel et al. 2005). This DAG-induced Ca2+ entry was blocked by Gd3+. However, the broad spectrum Ca2+ channel antagonist Gd3+ also inhibits the Na+/Ca2+ exchanger (Zhang and Hancox 2000) and the OAG-induced elevation of Na+. PKC, a major downstream DAG effector, can increase the cytosolic concentration of Ca2+ in some cell types (Albert et al. 1987; Khoyi et al. 1999; Murthy et al. 2000; Rosado and Sage 2000; Albert and Large 2002). In contrast to OAG or SAG, the PKC activator PMA failed to trigger any Ca2+ response. Furthermore, stimulating or inhibiting PKC activity had no effect on the DAG-induced Ca2+ entry. Taken together, these results favour the existence of cation channels activated by DAG in a PKC-independent manner.
TRPC are Gd3+- or SKF-96365-sensitive plasma membrane proteins forming voltage-independent cation channels. Some isoforms constitute DAG-sensitive Ca2+-conducting channels. When heterogeneously expressed, homomeric TRPC3, TRPC6 or TRPC7 channels and heteromeric TRPC1–TRPC3, TRPC3–TRPC4 channels function as DAG-sensitive Ca2+-conducting channels (Hofmann et al. 1999; Lintschinger et al. 2000; Liu et al. 2005; Poteser et al. 2006). Such DAG-sensitive channels have been described in the neuronal cell lines PC12 and IMR-32 (Mwanjewe and Grover 2004; Nasman et al. 2006) as well as in neural cells like cortical astrocytes (Grimaldi et al. 2003; Beskina et al. 2007), vomeronasal neurons (Lucas et al. 2003), hippocampal neurons (Tai et al. 2008) and neural stem cells (Pla et al. 2005). TRPC3 (in rats) (Grimaldi et al. 2003) or TRPC6 channels (in mice) (Beskina et al. 2007) participate in the DAG-induced cytosolic Ca2+ changes in astrocytes but the Ca2+ entry occurs through TRPC2 channels in vomeronasal neurons (Lucas et al. 2003) and through TRPC6 channels in hippocampal neurons (Tai et al. 2008). Based on these findings, we suggest that TRPC channels or channels exhibiting TRPC-like properties are involved in the DAG-dependent Ca2+ entry of cortical neurons. Among the various TRPC isoforms described so far, only currents through TRPC6 channels are increased by FFA. This anti-inflammatory drug is a potent blocker of anion and cation channels including TRPC (Inoue et al. 2001). But heterogeneously expressed TRPC6 channels (in HEK cells) and native TRPC6 channels (in vascular smooth muscle cells) are up-regulated by FFA (Inoue et al. 2001; Jung et al. 2002). In smooth muscle cells, FFA inhibits native Ca2+-conducting channels having TRPC3- and TRPC7-like properties with an IC50 value of 2.45 μM (Peppiatt-Wildman et al. 2007) but increases currents through native TRPC6-like channels (Hill et al. 2006). On the other hand, based on their electrophysiological and pharmacological studies, Carter et al. (2006) concluded that TRPC6 was involved in the ADP-dependent cation influx of murine megakaryocytes (Carter et al. 2006). Interestingly, this response was strongly enhanced by FFA (Carter et al. 2006). Therefore, TRPC6 (Inoue et al. 2001; Jung et al. 2002) and TRPC6-like channels (Carter et al. 2006; Hill et al. 2006) appear as the only TRPC channels up-regulated by FFA. In E13 cortical neurons, the OAG-induced Ca2+ entry was enhanced by FFA. Furthermore, hyperforin which selectively activates TRPC6 channels without activating TRPC1, TRPC3, TRPC4 and TRPC5 (Leuner et al. 2007) mimics the action of OAG. Electrophysiological measurements showed that this antidepressant activated non-selective cation channels blocked by Gd3+ (not shown) and SKF-96365. This in line with a recent study showing that hyperforin selectively activates non-selective cation TRPC6 channels (Leuner et al. 2007). In addition, hyperforin-activated currents were increased by FFA.
On the other hand, if the DAG-sensitive channels of cortical neurons exhibit TRPC6-like characteristics, they however display properties that are not found in other cells expressing TRPC6 channels. For instance, in HEK cells over-expressing TRPC6 channels, disruption of lipids rafts abolishes Ca2+ entry through TRPC6 channels (Aires et al. 2006) whereas the same treatment has no effect in cortical neurons. It is however possible that native and over-expressed TRPC6 channels exhibit distinct properties as already shown for TRPC3 where the mode of regulation of this TRPC isoform critically depends on its level of expression (Putney 2004). Another property of the DAG-sensitive channels of cortical neurons is their insensitivity to the tyrosine kinase inhibitors PP2 and genistein. Indeed, the src tyrosine kinase inhibitor PP2 abolishes endogenous TRPC6-dependent Ca2+ entry in cardiac myocytes (Nishida et al. 2007) and in HEK cells over-expressing TRPC6 channels (Aires et al. 2006) but has no effect on cortical neurons. Of note, Kawasaki et al. (2006) also reported that TRPC6 channels were unaffected by the tyrosine kinase inhibitor genistein.
Although the molecular identity of the DAG-sensitive channels of cortical neurons is not firmly established we exclude TRPC3 as the main candidate. This is based on the experiments carried out with PMA and showing that, after a PMA treatment, OAG was still able to promote an entry of Ca2+. Indeed, PKC activation totally blocks TRPC3 in response to OAG (Trebak et al. 2003; Venkatachalam et al. 2003; Kwan et al. 2006). A key issue concerns the characterization of the physiological activator(s) of these channels. None of the neurotransmitter receptor agonist tested (bethanechol, α-methyl-5-hydroxytryptamine, phenylephrine and histamine) had an effect. Although a clear understanding of the signalling pathway controlling the DAG-activated cation channels remains unclear as well as their exact molecular identity, we provide experimental evidence for the existence of functional second messenger-operated cationic channels in cortical neurons from E13 mouse embryos. Inositol 1,4,5-trisphosphate and DAG are second messengers playing important roles in cell signalling. Inositol 1,4,5-trisphosphate links cell surface receptors and Ca2+ signalling whereas DAG is the physiological activator of protein kinase C and thus controls protein phosphorylation. This latter process is regarded as one of the most important molecular mechanisms by which extracellular signals produce their biological responses (Walaas and Greengard 1991). As already shown, DAG also regulates in a PKC independent manner the activity of some plasma membrane ion channels (Hofmann et al. 1999; Lintschinger et al. 2000; Poteser et al. 2006). By controlling the activity of various ion channels and the phosphorylation of a plethora of proteins, DAG is as a second messenger with a widespread biological importance.