Because only 10% of the ingested dye is absorbed in the intestines, concentrations high enough to affect Panx1 channels are not to be expected if the acceptable daily intake value is adhered to

Because only 10% of the ingested dye is absorbed in the intestines, concentrations high enough to affect Panx1 channels are not to be expected if the acceptable daily intake value is adhered to. contrast to other P2X7R antagonists had no significant inhibitory effect on Panx1 channels. Based on its selective action, Rabbit Polyclonal to HDAC5 (phospho-Ser259) BB FCF can be added to the repertoire of drugs to study the physiology of Panx1 channels. Furthermore, because Panx1 channels appear to be involved directly or indirectly through P2X7Rs in several disorders, BB FCF and FTY720 (Fingolimod) derivatives of this safe food dye should be given serious consideration for pharmacological intervention of conditions such as acute Crohns disease, stroke, and injuries to the central nervous system. INTRODUCTION Purinergic receptors, specifically the P2X7 receptor (P2X7R), have been recognized as a potential site of intervention for the treatment of several neurological disorders including spinal cord injury, Huntingtons disease, and other neurodegenerative diseases involving neuroinflammation (Daz-Hernndez et al., 2009; Peng et al., 2009; Takenouchi et al., 2010; Lopat? et al., 2011; Traini et al., 2011; Arbeloa et al., 2012; Chu et al., 2012; Iriyama et al., 2012; Iwamaru et al., 2012; Kimbler et al., 2012). The P2X7R is usually a ligand-operated ion channel with high permeability to small cations (North and Barnard, 1997; North, 2002). In a second incarnation, the P2X7R also can form a large pore, which allows the flux of larger molecules such as the dye YoPro. Whether the large pore formation is an inherent property of the P2X7R protein or whether a pore-forming protein is usually associated with the P2X7R is usually a matter of debate (Pelegrin and Surprenant, 2006; Locovei et al., 2007; Chaumont and Khakh, 2008). Several drugs interact with the P2X7R and block its channel and large pore activity with high efficacy and good selectivity among purinergic receptors. This includes Brilliant Blue G (BBG), a dye widely used as a stain for protein assays. Depending on the species, BBG inhibits the P2X7R with an IC50 of 10 nM (rat) or 265 nM (human), while requiring 100C1,000 times higher concentrations to inhibit other P2X receptors (Jiang et al., 2000). BBG exhibits some structural similarity to Brilliant Blue FCF (BB FCF), the safe food dye FD&C Blue No. 1. Many publications point to this similarity with the salient implication that BB FCF acts around the P2X7R in the same way as BBG. A Medline search with terms P2X7 and BB FCF or other names of the dye such as Erioglaucine yields in excess of 100 references. Yet most (if not all) fail to contain data on effects of the dye on P2X7-mediated membrane currents. Instead, these papers describe effects of BBG and refer to the structural similarity between BB FCF and BBG. To our knowledge, the only study to actually test BB FCF for effects on any membrane channel is usually that of FTY720 (Fingolimod) Jo and Bean (2011), who exhibited that BBG with an IC50 of 2 M inhibits voltage-gated sodium channels, and that BB FCF requires a considerably higher concentration to affect these channels. The P2X7R can act in concert with the ATP release channel pannexin 1 (Panx1; Pelegrin and Surprenant, 2006; Locovei et al., 2007). A plausible role for Panx1 in that collaboration is usually that of an amplifier, boosting the ligand concentration at the receptor. This potentially dangerous positive feedback loop is usually counteracted by a negative control mechanism in which ATP binds to a site around the extracellular surface of Panx1, inhibiting the channel activity of the protein (Qiu and Dahl, 2009; Qiu et al., 2012). The ATP-binding sites on Panx1 and P2X7R are comparable in that positively charged amino acids are involved in forming the binding site and FTY720 (Fingolimod) that several ligands to the receptor, including FTY720 (Fingolimod) agonists such as BzATP and antagonists such as BBG, inhibit the Panx1 channel. The major difference between the binding sites is usually their affinity, with Panx1 using a considerably lower affinity to ATP and other ligands than P2X7R (Qiu et al., 2012). In testing whether this relationship holds up also for other P2X7R ligands, we investigated the effect of BB FCF. Surprisingly, the food dye did not affect P2X7R-mediated currents. Instead, BB FCF inhibited Panx1 channel currents in submicromolar concentrations. MATERIALS AND METHODS Preparation of oocytes Preparation of oocytes and electrophysiological recording were performed as described previously (Dahl, 1992; Dahl and Pfahnl, 2001). Mouse Panx1 was provided by R. Dermietzel (University of Bochum, Bochum, Germany), and connexin 46 (Cx46) was obtained from D.L. Paul (Harvard University, Cambridge, MA). Human P2X7R was provided by A. Surprenant (University of.