Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63

Masahiro Ikeda, Eric Beitz, David Kozono, William B. Guggino, Peter Agre, Masato Yasui

Research output: Contribution to journalArticle

151 Citations (Scopus)

Abstract

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; PNO3/PCl >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.

Original languageEnglish
Pages (from-to)39873-39879
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number42
DOIs
Publication statusPublished - 2002 Oct 18
Externally publishedYes

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Aquaporin 6
Threonine
Linings
Nitrates
Anions
Aquaporins
Fluorescence
Cells
Cell membranes
Ions
Cell Membrane
Proteins
Isoleucine
Xenopus laevis
Ion Channels
Permeation
Oocytes
Rats
Permeability
Kidney

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63. / Ikeda, Masahiro; Beitz, Eric; Kozono, David; Guggino, William B.; Agre, Peter; Yasui, Masato.

In: Journal of Biological Chemistry, Vol. 277, No. 42, 18.10.2002, p. 39873-39879.

Research output: Contribution to journalArticle

Ikeda, Masahiro ; Beitz, Eric ; Kozono, David ; Guggino, William B. ; Agre, Peter ; Yasui, Masato. / Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 42. pp. 39873-39879.
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