Abstract
The transport of CO2 across biomembranes in plant cells is essential for efficient photosynthesis. Some aquaporins capable of CO2 transport, referred to as 'COOporins', are postulated to play a crucial role in leaf CO2 diffusion. However, the structural basis of CO2 permeation through aquaporins remains largely unknown. Here, we show that amino acids in loop C are critical for the CO2 permeability of Arabidopsis thaliana PIP2 aquaporins. We found that swapping tyrosine and serine in loop C to histidine and phenylalanine, which differ between AtPIP2;1 and AtPIP2;3, altered CO2 permeability when examined in the Xenopus laevis oocyte heterologous expression system. AlphaFold2 modelling indicated that these substitution induced a conformational shift in the sidechain of arginine in the aromatic/arginine (ar/R) selectivity filter and in lysine at the extracellular mouth of the monomeric pore in PIP2 aquaporins. Our findings demonstrate that distal amino acid substitutions can trigger conformational changes of the ar/R filter in the monomeric pore, modulating CO2 permeability. Additionally, phylogenetic analysis suggested that aquaporins capable of dual water/CO2 permeability are ancestral to those that are water-selective and CO2-impermeable, and CO2-selective and water impermeable.