The hidden cation-selective pore in ion-conducting aquaporin OsPIP2;4 from rice

Ion-conducting aquaporins (icAQPs) transport ions as well as water. Although the molecular mechanism of how AQPs establish selective permeability for water molecules is well understood, the ion-transporting mechanism in icAQPs has not yet been fully elucidated. In this study, we investigated the molecular mechanism of cation transport in OsPIP2;4, an icAQP in rice, by homology modeling and the electrophysiological analysis using Xenopus laevis oocytes. Water and ion transport assays using OsPIP2;4 T227M and G278K mutants strongly suggested that water- and cation-transporting pathways are independent of each other. Data from amino acid substitutions V54I and A143G in OsPIP2;4 led to the identification of a novel hidden pathway for cation transport located on the side surfaces of the tetramer channel, where two protomers are in contact, which is distinct from conventional monomeric pores and the tetrameric central pore in AQPs. Moreover, the present results provide the possibility that this hypothetical hidden pore also functions in the barley icAQP HvPIP2;8. The overall structure of this novel pathway appears to differ from the structure of general cation channels. However, the arrangement of hydrophilic amino acids at the entrance of the pathway of OsPIP2;4 was found to be comparable to that of some cation channels, which implies that the molecular mechanism of dehydration of hydrated ions might resemble that of the channels. Although direct structural evidence is needed to confirm the proposed pathway, the present study can be a stepping stone toward unraveling the mechanism of dual water and ion transport through icAQPs in plants.