Subtype-specific potentiation of otopetrin proton channels by Zn2+ and its structural basis.

Abstract

Otopetrin proteins (OTOPs) form proton-selective ion channels that are widely distributed throughout the body. In vertebrates, OTOP1 functions as the sour receptor while the physiological functions of related proteins, OTOP2, and OTOP3, are not yet known. To better understand how these channels may contribute to physiology, we sought to describe their basic functional properties. Previous work showed that both OTOP1 and OTOP3 are activated by protons while OTOP2 is constitutively active and all three channels are inhibited by Zn2+. Here we show that Zn2+ functions as a potent activator of murine OTOP3. Pre-exposure for 1-64s to Zn2+ speeds up the activation and increases the magnitude of OTOP3 currents by as much as 10-fold. In contrast, OTOP2 currents are insensitive to potentiation by Zn2+ while OTOP1 currents are potentiated to a lesser degree. Testing of chimeras between OTOP3 and OTOP2 showed that swapping the extracellular tm 11-12 linker between the channels was sufficient to eliminate Zn2+ potentiation of OTOP3 and confer Zn2+ potentiation on OTOP2. Site-directed mutagenesis revealed that H531 within the tm 11-12 linker is essential for potentiation of OTOP3 by Zn2+ and likely contributes to its binding site. Kinetic modeling of the data is consistent with Zn2+ stabilizing the open state of the channel, possibly competing with H+ for activation of the channels. These results point to the 11-12 linker as part of the gating apparatus of OTOP channels and a target for drug discovery. Zn2+ is an essential micronutrient, and its regulation of OTOP channels will undoubtedly have important physiological sequelae yet to be discovered.