Wang J et al. (2013), The food dye FD&C Blue No. 1 is a selective inh...

XB-ART-53494

J Gen Physiol 2013 May 01;1415:649-56. doi: 10.1085/jgp.201310966.

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Wang J , Jackson DG , Dahl G .

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The food dye FD&C Blue No. 1 (Brilliant Blue FCF [BB FCF]) is structurally similar to the purinergic receptor antagonist Brilliant Blue G (BBG), which is a well-known inhibitor of the ionotropic P2X7 receptor (P2X7R). The P2X7R functionally interacts with the membrane channel protein pannexin 1 (Panx1) in inflammasome signaling. Intriguingly, ligands to the P2X7R, regardless of whether they are acting as agonists or antagonists at the receptor, inhibit Panx1 channels. Thus, because both P2X7R and Panx1 are inhibited by BBG, the diagnostic value of the drug is limited. Here, we show that the food dye BB FCF is a selective inhibitor of Panx1 channels, with an IC50 of 0.27 µM. No significant effect was observed with concentrations as high as 100 µM of BB FCF on P2X7R. Differing by just one hydroxyl group from BB FCF, the food dye FD&C Green No. 3 exhibited similar selective inhibition of Panx1 channels. A reverse selectivity was observed for the P2X7R antagonist, oxidized ATP, which in contrast to other P2X7R antagonists had no significant inhibitory effect on Panx1 channels. Based on its selective action, 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 derivatives of this "safe" food dye should be given serious consideration for pharmacological intervention of conditions such as acute Crohn's disease, stroke, and injuries to the central nervous system.

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Species referenced: Xenopus laevis
Genes referenced: gja3 p2rx7 panx1 slco1a2

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	Figure 1. BzATP- induced currents in oocytes expressing the human P2X7R. The membrane potential was clamped at â60 mV, and brief 10-mV pulses were applied at a rate of 0.1 Hz for assessment of changes in membrane conductance. The currents and conductance changes induced by 100 ÂµM BzATP were either sustained (A) or diminished (B) over time, despite the presence of the stimulant depending on the batch of oocytes. BzATP-induced currents in oocytes were inhibited by BBG but not by BB FCF (CâE). Experimental conditions were the same as in A, except the inhibitors BB FCF and BBG were included. (C) In the presence of 100 ÂµM BB FCF, membrane currents of significant amplitude were induced by BzATP, which were attenuated by 10 ÂµM BBG. (D) When applied in the presence of BzATP, the induced currents were minimally affected by 100 ÂµM BB FCF. (E) Quantitative analysis of membrane currents induced by BzATP in the presence of 100 ÂµM BB FCF or of various concentrations of BBG. Means Â± SD are plotted, and n is indicated.
	Figure 2. Dose-dependent inhibition of Panx1 currents by BB FCF. The membrane potential was clamped at â60 mV, and brief +60-mV pulses were applied at a rate of 0.1 Hz to open Panx1 channels. (A) BB FCF attenuated the Panx1-mediated currents in a dose-dependent fashion. (B) Doseâresponse relationship of inhibition of membrane currents by BB FCF. Means Â± SD (n = 4) are plotted. (C) Uninjected control oocytes subjected to the same pulse protocol exhibited small currents unaffected by 10 ÂµM BB FCF and by 10 ÂµM Fast Green FCF.
	Figure 3. PubChem structures of dyes used in this study. The food dyes BB FCF and Fast Green FCF are structurally identical except for one OH group. BBG is structurally related to the food dyes but exhibits clear differences.
	Figure 4. KGlu-induced ATP release from oocytes expressing Panx1 was inhibited by BB FCF. ATP release was determined with the luciferase assay, with which the dye interferes. A correction factor for this interference was determined and used for plotting the data.
	Figure 5. Failure of BB FCF to affect connexin channels. Two connexins, Cx32E143 and Cx46, forming open âhemichannelsâ at regular calcium ion concentrations in oocytes expressing them, were tested. The membrane potential was clamped at â50 mV, and brief pulses to 0 mV were applied at a rate of 0.1 Hz to open the connexin channels. 100 ÂµM BB FCF did not affect the currents carried by either connexin. The traces are representative of four trials for each connexin on different oocytes.
	Figure 6. Effect of various Panx1 inhibitors in alanine replacement mutants lacking the feedback inhibition by ATP. The Panx1 inhibitors carbenoxolone, BB FCF, Fast Green FCF, and probenecid were tested in wild-type Panx1 and various alanine replacement mutants using the same protocol as in Fig. 2. The inhibition by probenecid was almost abolished in Panx1 W74A. Carbenoxolone and Fast Green FCF inhibited the currents effectively in all mutants. BB FCF inhibition was attenuated in all mutants except Panx1 I247A. Means Â± SD are plotted. n = 5 for Panx1 I247A and L266A, and n = 4 for all other points.
	Figure 7. Effect of oATP on Panx1 currents and P2X7R-mediated currents induced by 100 ÂµM BzATP. con, voltage-induced Pannx1 currents or BzATP-induced P2X7R currents in the absence of oATP. Oocytes expressing Panx1 or P2X7R were preincubated with 100 ÂµM oATP for 2â3 h before measurements of membrane currents. oATP only attenuated BzATP-induced currents in P2X7R-expressing oocytes. Means Â± SD are plotted, and n is indicated.

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