Structural and functional characterization of an otopetrin family proton channel

Abstract

The otopetrin (OTOP) proteins were recently characterized as proton channels. Here we present the cryo-EM structure of OTOP3 from Xenopus tropicalis (XtOTOP3) along with functional characterization of the channel. XtOTOP3 forms a homodimer with each subunit containing 12 transmembrane helices that can be divided into two structurally homologous halves; each half assembles as an α-helical barrel that could potentially serve as a proton conduction pore. Both pores open from the extracellular half before becoming occluded at a central constriction point consisting of three highly conserved residues – Gln232/585-Asp262/Asn623-Tyr322/666 (the constriction triads). Mutagenesis shows that the constriction triad from the second pore is less amenable to perturbation than that of the first pore, suggesting an unequal contribution between the two pores to proton transport. We also identified several key residues at the interface between the two pores that are functionally important, particularly Asp509, which confers intracellular pH-dependent desensitization to OTOP channels.

Structures of the otopetrin proton channels Otop1 and Otop3

“…Here we have described cryo-EM structures of Otop1 and Otop3 in lipidic nanodiscs, elucidating the topology and dimeric organization of Otopetrins in a native-like bilayer environment. We also note that a recent study33 showed that detergent-solubilized Otop3 from Xenopus tropicalis adopts the same overall homodimeric architecture as the structures we have described here.…”

Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels

“…The unusual response properties of OTOP2 raise the question of whether indeed OTOP2 is selective for protons. OTOP1 is highly selective for H + over Na + , by a factor of at least 10 5 fold (Tu et al 2018), and currents carried by OTOP3 follow the expectations for a proton-selective current, such as a shift in the reversal potential that follows the equilibrium potential for the H + ion (Tu et al 2018; Chen et al 2019). OTOP2 is known to permeate protons (Tu et al 2018), but selectivity for protons was not previously measured.…”

“…The recent cryo-EM structures of OTOP1 and OTOP3 channels have revealed that the channels adopt a novel fold, with twelve transmembrane alpha-helices organized into two structurally homologous six-helix domains (N and C domains) (Saotome et al 2019; Chen et al 2019). Thus, as a dimer, the channels adopt a pseudo-tetrameric structure.…”

“…HEK293 cells were purchased from ATCC (CRL-1573). PAC-KO cells were a kind gift from Dr. Zhaozhu Qiu (Yang, Chen, et al 2019). The cells were cultured in a humidified incubator at 37C in 5% CO2 and 95% O2 using a high glucose DMEM (ThermoFisher) containing 10% fetal bovine serum (Life Technology) and 1% Penicillin-streptomycin antibiotics.…”

How low can we go? Structure determination of small biological complexes using single-particle cryo-EM

“…It is perhaps unsurprising, then, that membrane proteins stabilized in detergents or lipid nanodiscs comprise over 60% of all high-resolution sub-200 kDa structures deposited into the EMDB to date, with nearly all entries obtained within the past two years alone. Among these were de novo structures of the human lipid exporter ABCB4 [54], the otopetrin proton channels OTOP1 [55] and OTOP3 [55,56], several members of the TMEM16 scramblase family [40,57,58], as well as of the structurally homologous OSCA mechanosensitive ion channel family [46,59]. It is interesting to note that the majority of structures were obtained using a microscope equipped with an energy filter, which may have provided some gain in SNR particularly around the transmembrane region, which is surrounded by disordered detergent or lipid molecules.…”