MRS5474 inhibited GABA uptake through an adenosine A3 receptor
GABA transporters (GAT), in particular the GABA transporter type-1 (GAT-1) are well-known targets for antiseizure medications (Meldrum and Chapman, 1999; Sills and Rogawski, 2020). Importantly, recent evidence suggests that GAT-1 inhibitors not only inhibit seizures but may also halt epileptogenesis (Javaid et al., 2023). We therefore hypothesized that MRS5474 could act by inhibiting GAT-1 mediated GABA transport. Since GAT-1 is present in nerve endings and astrocytes, we tested the effect of MRS5474 in slices, which enabled us to evaluate the effect upon GABA transport independently of its specific location. In slices in the presence of MRS5474 (50 nM,) the GAT-1 mediated uptake of [3H]GABA was significantly lower than under control conditions (% inhibition: 47.3±6.5%, n=15, Figure 5A and C). This effect of MRS5474 at 50nM was not maximal since higher concentrations caused stronger inhibition (120nM: 89±5.9%, n=6; 250nM: 96±2.3%, n=4) of GAT-1 mediated [3H]GABA uptake. Unexpectedly, the presence of the A1R antagonist, DPCPX, used at a concentration (50 nM) 100 times higher than its Ki value for A1R (Lohse et al., 1987), and added 30 minutes before MRS5474, did not prevent the action of MRS5474 (n=4, P>0.05, Figure 5D ). In contrast, the inhibition was fully prevented in experiments where MRS1523 (10 µM), an A3R antagonist (Li et al., 1998), was added to the incubation media before MRS5474 (n=5, P<0.05, Figure 5D ), indicating that the inhibitory action of MRS5474 upon GAT-1 activity is mediated by A3R. Neither DPCPX (50nM) nor MRS1523 (10µM) significantly (P>0.05) affected GAT-1 activity as compared with controls (no drug) in the same experiments (Figure 5 E-F). To further assess the involvement of A3R upon GAT-1 mediated GABA uptake, we tested the action of a selective A3R agonist, MRS5698 (Ki ≈3nM in human or mouse, A2AR; Tosh et al., 2012b). As shown in Figure 5 (B–D ), in the presence of MRS5698 (100 nM), GAT-mediated GABA uptake was significantly lower than in its absence (% inhibition: 47.7±11.1%, n=8, P<0.05). The mimicry of the effect of MRS5474 by a selective A3R agonist, together with the blockade of the effect of MRS5474 by an A3R antagonist but not by a selective A1R antagonist, strongly suggest that the effect of MRS5474 is mediated by A3R rather than A1R.