Concerted action of two cation filters in the aquaporin water channel

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water‐specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H + ≫K + >Rb + >Na + >Cs + >Li + . Contrary to theoretical models, we found that electrostatic repulsion at the central asn‐pro‐ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali‐leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali‐tight but solute‐unselective NPA region as a feature of primordial channels and the proton‐tight and solute‐selective ar/R constriction variants as later adaptations within the AQP superfamily.