Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63.
Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg2+ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH-induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl- revealed high nitrate permeability even at pH 7.4; P(NO3)/P(Cl) > 9.8. Site-directed mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO3-/Cl- selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO3-/Cl- mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport.
Ikeda
Masahiro
M
Departments of Physiology, Biological Chemistry, Medicine, and Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Beitz
Eric
E
Kozono
David
D
Guggino
William B
WB
Agre
Peter
P
Yasui
Masato
M
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000737:chemistry* / Q000502:physiology*; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007532:Isoleucine / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009566:Nitrates / Q000737:chemistry*; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012984:Software; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D014982:Xenopus laevis
C496023:AQP2 protein, human; C496025:Aqp2 protein, rat; C496043:Aqp6 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D007477:Ions; D008164:Luminescent Proteins; D009566:Nitrates; D007532:Isoleucine; D049452:Green Fluorescent Proteins; D013912:Threonine; D014443:Tyrosine