Cibacron blue 3G-A is a novel inhibitor of Otopetrin 1 (OTOP1), a proton channel

Abstract

Otopetrin 1 (OTOP1) is a proton (H⁺) channel which detects acidic stimuli in sour taste receptor cells and plays some sort of role in the formation of otoconia in the inner ear. Although it is known that zinc ion (Zn²⁺) inhibits OTOP1, Zn²⁺ requires high concentrations (mM order) to inhibit OTOP1 sufficiently, and no other inhibitors have been found. Therefore, to identify a novel inhibitor, we screened a chemical library (LOPAC¹²⁸⁰) by whole-cell patch clamp recordings, measuring proton currents of heterologously-expressed mouse OTOP1. From the screening, we found that reactive blue 2 inhibited OTOP1 currents. Further evaluations of three analogues of reactive blue 2 revealed that cibacron blue 3G-A potently inhibited OTOP1 currents. Cibacron blue 3G-A inhibited OTOP1 currents in a concentration-dependent manner, and its 50% inhibitory concentration (IC50) and the Hill coefficient were 5.0 μM and 1.1, respectively. The inhibition of OTOP1 currents by cibacron blue 3G-A was less affected by extracellular anion compositions, membrane potentials, and low pH than the inhibition by Zn²⁺. These results suggest that the inhibition of OTOP1 by cibacron blue 3G-A is neither likely to be a pore-blocking inhibition nor a competitive inhibition. Furthermore, our findings revealed that cibacron blue 3G-A can be used as a novel inhibitor of OTOP1 especially under the conditions in which OTOP1 activity is evaluated such as low pH.

Keywords: Cibacron blue 3G-A; OTOP1; Otopetrin1; Proton channel; Reactive blue 2.