Fig. 1: Characterization of the compounds MFaN, HIMOP, and B2FAMP as positive allosteric modulators of the Otop1 channel.
From: Preferential allosteric modulation of Otop1 channels by small molecule compounds
A Typical pH 5.5-activated currents of mouse Otop1 (mOtop1) channels heterologously expressed in HEK293T cells (n = 5). B Structures of MFaN, HIMOP, and B2FAMP (upper panels) and typical current traces showing that these compounds (at 10 μM concentration) potently potentiated pH 5.5-activated mOtop1 currents (lower panels). ΔI1 and ΔI2 indicate the currents immediately before, and the peak current after compounds treatment, respectively (n = 5). C Dose-response relationships of compounds potentiating the acid-activated mOtop1 currents, with the EC50s being determined as 10.3 ± 2.6 μM, 16.8 ± 6.5 μM, and 12.2 ± 3.4 μM for MFaN, HIMOP, and B2FAMP at pH 5.5, and 8.9 ± 4.7 μM for MFaN at pH 6.0 (n = 5). The maximum ΔI2/ΔI1 ratio of other groups were compared to that of the MFaN/pH 5.5 group. D Typical mOtop1 currents activated by alkali/acid stimuli at various pH levels, under conditions without (middle panel; 0.1% DMSO) and with co-administration of MFaN (lower panel; 10 μM), upper panel demonstrates currents recording from untransfected cells (n = 6–15). E Summary ΔI2/ΔI1 ratio in (D) at various pHs, showing MFaN potently potentiated pH 4.5–6.0 acid-activated, but not alkali or pH 4.0 acid-activated mOtop1 currents, when compared to the pH 3.5 group in which the compound did not affect the mOtop1 currents. Each dot represents an independent experimental cell (n = 6–15). F, G Typical current traces (F) and bar statistics (G) showing both B2FAMP and HIMOP markedly potentiated pH 5.5 but not pH 3.5-activated mOtop1 currents (n = 5–8). H Current density (pA/pF) of mOtop1 channels activated by alkali/acid stimuli at different pH levels is shown for conditions without MFaN (black dots) and with co-administration of 10 μM MFaN (red dots). The data demonstrate that MFaN significantly potentiated mOtop1 currents at pH levels between 4.5 and 6.0, but not at other pH levels. Each dot represents an independent experimental cell (n = 5–15). I Each representative trace illustrates two mOtop1 currents in the same recording cell sequentially evoked by stimuli of pH 6.0 alone (Ip1) and pH 6.0 with MFaN (of different concentrations; Ip2) (n = 4–7). J Dose-response relationships of MFaN potentiating pH 6.0-activated mOtop1 currents in (I), with the EC50 being determined as 9.2 ± 0.6 μM (n = 4–7). In these figure panels, currents were recorded at 0 mV for pH values 9.0 and 9.5, and at −80 mV for all other pH values; dotted line indicates the baseline current, and the solution perfusion protocols are as illustrated; Data are presented as MEAN ± SEM. Statistics: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ONE-WAY ANOVA with post-hoc Dunnett analysis in panels (C, E), and unpaired t-test in panels (G, H).
