Characterization of the antagonist actions of 5-BDBD at the rat P2X4 receptor

Abstract

P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC₅₀ of 0.75 μM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 μM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 μM concentration but not 0.75 μM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.

Keywords: 5-BDBD; Long-term potentiation; P2X4 receptor; Purinergic signaling.

Figure 1.. 5-BDBD inhibits rP2X4R-mediated currents.

A. Representative recording from a single HEK293 cell expressing the rP2X4R. Currents were gated by a 5 s application of 10 µM ATP, voltage membrane was set to −60 mV. 5-BDBD was pre-applied for 2 min and then co-applied with ATP at the concentrations indicated; washout between ATP pulses was 4 minutes. B. Summary of the 5-BDBD concentration-response protocols performed on rP2X4R expressing cells and activated by 10 µM ATP. Curves were normalized to the response obtained by ATP alone at the beginning of the experiments. Symbols represent the mean values, and bars indicate SE values from n = 7.

Figure 2.. A rightward shift in the ATP EC₅₀ by 5-BDBD.

A. Representative recordings from rP2X4R-HEK293 cells of ATP induced currents in the absence (upper panel) and in the presence of 1 µM 5-BDBD, pre-applied for 2 minutes and then co-applied with ATP (lower panel). Voltage membrane was held at −60 mV. B. ATP concentration-response curves in the absence (black circles) and presence (open squares) of 1 µ 5-BDBD. Curves were normalized against the maximal response obtained in each condition. Dotted lines represent the EC₅₀ values, 4.7 ± 1.8 µM (n = 7) in the absence and 15.9 ± 3.9 µM (n = 4) in the presence of 1 µM 5-BDBD.

Figure 3.. Specificity of 5-BDBD among rat P2XRs.

A. Representative recordings of HEK293 cells expressing the P2X2aR, P2X2bR, P2X3R or P2X7R in the absence or in the presence of 10 µM 5-BDBD (pre-applied for 2 minutes and then co-applied with ATP). The ATP concentrations used were 1, 10 and 1000 µM A P for the P2X3R, P2X2R and P2X7R, respectively. Voltage membrane was held at −60 mV. B. Summary of the results obtained with 5-BDBD. ^*, p < 0.05; ^**, p < 0.01, as compared with the response obtained with ATP alone (inside bars) or comparing between P2XRs (Mann-Whitney test).

Figure 4.. Effects of 5-BDBD on LTP.

A and B. Representative Western blots of P2X4R (A and B) or P2X1R and P2X3R (B) in synaptic fractions obtained from rat hippocampus (right panel). H: Homogenate, S: supernatant, PSD: Postsynaptic densities. As P2X4R positive control, transfected HEK cells were used. C. LTP curves obtained from rat hippocampal slices incubated for 1 hour with 10 µM 5-BDBD in aCSF (grey squares, n=20 slices, 6 rats) or aCSF alone (white circles, n = 16 slices, 5 rats). D. LTP curves obtained from slices incubated by 1 hour with 3 µM PPADS (dark grey squares, n=8 slices, 3 rats) or 100 nM AF-353 (light grey diamonds, n=9 slices, 6 rats) and the corresponding control experiments (white circles, n=18 slices, 6 rats). TBS was applied after 20 minutes of stable baseline. %fEPSP: % field Excitatory Postsynaptic Potential, TBS: Theta burst stimulation E. Late phase-LTP quantification of curves obtained from experiments shown in C and D. Mean %fEPSP were obtained from each curve taking points from 40 to 60 minutes and compared using t-student test. ^***p <0.001.