Characterization of aquaporin-driven hydrogen peroxide transport.

Aquaporins are membrane-intrinsic proteins initially defined as water (HO) channels in all organisms and subsequently found to have multiple substrate specificities, such as hydrogen peroxide (HO). HO 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 HO 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 HO 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 HO 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 Cd 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 HO and HO, but have different efficiencies for various specificities. Notably, all three homologues expedite HO transport equally well while the plant aquaporins are more permeable to HO than hAQP1. Comparison of the structures indicates that the observed variations in HO and HO 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.