Structure-function analysis of plant aquaporin AtPIP2;1 gating by divalent cations and protons.

ABSTRACT

Water channel proteins, AQPs (aquaporins), of the PIP (plasma membrane intrinsic protein) subfamily, provide a means for fine and quick adjustments of the plant water status. A molecular model for gating of PIPs by cytosolic protons (H(+)) and divalent cations was derived from the atomic structure of spinach SoPIP2;1 (Spinacia oleracea PIP2;1) in an open- and a closed-pore conformation. In the present study, we produced the Arabidopsis AtPIP2;1 (Arabidopsis thaliana PIP2;1) homologue in Pichia pastoris, either WT (wild-type) or mutations at residues supposedly involved in gating. Stopped-flow spectrophotometric measurements showed that, upon reconstitution in proteoliposomes, all forms function as water channels. The first functional evidence for a direct gating of PIPs by divalent (bivalent) cations was obtained. In particular, cadmium and manganese were identified, in addition to calcium (Ca(2+)) and H(+) as potent inhibitors of WT AtPIP2;1. Our results further show that His(199), the previously identified site for H(+) sensing, but also N-terminal located Glu(31), and to a lesser extent Asp(28), are involved in both divalent-cation- and H(+)-mediated gating. In contrast, mutation of Arg(124) rendered AtPIP2;1 largely insensitive to Ca(2+) while remaining fully sensitive to H(+). The role of these residues in binding divalent cations and/or stabilizing the open or closed pore conformations is discussed.