Characterization of aquaporin-driven hydrogen peroxide transport

Abstract

Aquaporins are membrane-intrinsic proteins initially defined as water (H₂O) channels in all organisms and subsequently found to have multiple substrate specificities, such as hydrogen peroxide (H₂O₂). H₂O₂ is a signaling molecule that partakes in immune responses where its transport is mediated by aquaporins. To shed further light on the molecular basis of the aquaporin function in H₂O₂ transport, we have characterized an Arabidopsis thaliana aquaporin, AtPIP2;4, recombinantly produced to high yields in Pichia pastoris. Here, we present a newly established assay that allows detection of H₂O₂ transport by purified aquaporins reconstituted into liposomes, enabling us to compare aquaporin homologues with respect to substrate specificity. To get additional insight into the structural determinants for aquaporin-mediated H₂O₂ transport, we solved the 3D-structure of AtPIP2;4 to 3.7 Å resolution and found structural identity to the water channel from spinach (SoPIP2;1), with the difference that Cd²⁺ cation is not required to retain the closed conformation. The transport specificities of the two plant aquaporins were compared to a human homologue, AQP1. Overall, we conclude that AtPIP2;4, SoPIP2;1 and hAQP1 are all transporters of both H₂O and H₂O₂, but have different efficiencies for various specificities. Notably, all three homologues expedite H₂O transport equally well while the plant aquaporins are more permeable to H₂O₂ than hAQP1. Comparison of the structures indicates that the observed variations in H₂O and H₂O₂ transport cannot be explained by differences in the monomeric pore. Possibly, the determinants for transport specificities reside in the flexible domains outside the membrane core of these channels.