Increased Permeability of the Aquaporin SoPIP2;1 by Mercury and Mutations in Loop A.
Aquaporins (AQPs) also referred to as Major intrinsic proteins, regulate permeability of biological membranes for water and other uncharged small polar molecules. Plants encode more AQPs than other organisms and just one of the four AQP subfamilies in Arabidopsis thaliana, the water specific plasma membrane intrinsic proteins (PIPs), has 13 isoforms, the same number as the total AQPs encoded by the entire human genome. The PIPs are more conserved than other plant AQPs and here we demonstrate that a cysteine residue, in loop A of SoPIP2;1 from Spinacia oleracea, is forming disulfide bridges. This is in agreement with studies on maize PIPs, but in contrast we also show an increased permeability of mutants with a substitution at this position. In accordance with earlier findings, we confirm that mercury increases water permeability of both wild type and mutant proteins. We report on the slow kinetics and reversibility of the activation, and on quenching of intrinsic tryptophan fluorescence as a potential reporter of conformational changes associated with activation. Hence, previous studies in plants based on the assumption of mercury as a general AQP blocker have to be reevaluated, whereas mercury and fluorescence studies of isolated PIPs provide new means to follow structural changes dynamically.
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University Lund, Sweden.
Andreas
A
Kirscht
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University Lund, Sweden.
Sabeen
S
Survery
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University Lund, Sweden.
Per
P
Kjellbom
Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University Lund, Sweden.
Urban
U
Johanson
Spinacia oleracea; aquaporin; major intrinsic protein; tryptophan fluorescence; water channel