Structure-function analysis of plant aquaporin AtPIP2;1 gating by divalent cations and protons.
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.
Verdoucq
Lionel
L
Biochimie et Physiologie Moleculaire des Plantes, Institut de Biologie Integrative des Plantes, UMR 5004 CNRS, UMR 0386 INRA/Montpellier SupAgro/Universite Montpellier 2, F-34060 Montpellier Cedex 1, France. verdoucq@supagro.inra.fr
Grondin
Alexandre
A
Maurel
Christophe
C
D020346:Aquaporins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D008958:Models, Molecular; D010843:Pichia / Q000378:metabolism; D011487:Protein Conformation; D011510:Proteolipids / Q000378:metabolism; D011522:Protons; D013329:Structure-Activity Relationship; D014867:Water / Q000378:metabolism
D020346:Aquaporins; D029681:Arabidopsis Proteins; D002413:Cations, Divalent; D011510:Proteolipids; D011522:Protons; C491680:plasma membrane intrinsic protein 2 Arabidopsis; C020485:proteoliposomes; D014867:Water; D002118:Calcium