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Chloride transport induced by the substitution of Asp555 with a Glu in the Na/HCO3 cotransporter SLC4A4 (NBCe1)
Author(s) -
Lee Soojung,
Yang Han Soo,
Nichols J. Wylie,
Rajbhandari Ira,
Choi Inyeong
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.800.3
Subject(s) - voltage clamp , chemistry , iodide , biophysics , reversal potential , chloride , cotransporter , intracellular , xenopus , transfection , biochemistry , patch clamp , microbiology and biotechnology , membrane potential , inorganic chemistry , biology , sodium , receptor , gene , organic chemistry
By structure/function analysis, we previously demonstrated that the substitution of Asp 555 in NBCe1 with a Glu (D555E) causes the mutant transporter to produce currents by other anions. In this study, we further characterized the chloride transport of D555E by using two‐electrode voltage clamp and fluorescence measurements. In Xenopus oocytes expressing D555E, the application of a series of Cl concentrations without CO 2 /HCO 3 produced outward currents in voltage‐clamp recordings. A similar application did not evoke outward currents in oocytes expressing wild type human NBCe1. The chloride currents induced by D555E were markedly diminished in CO 2 /HCO 3 solution, consistent with the previous finding that D555E produces Cl currents in the absence of HCO 3 . To address the issue of whether the chloride conductance is not derived from endogenous oocyte proteins but is intrinsic to the mutant transporter, we expressed NBCe1 and D555E in HEK293 cells and measured chloride transport by using the fluorescence dye 6‐methoxy‐N‐(‐sulphopropyl) quinolinium SPQ. The expression of the transporters in cells was confirmed by immunoblot and immunocytochemistry with the antibody specific to the C‐terminal end of the protein. SPQ‐loaded cells were washed with a solution containing nitrate, and intracellular SPQ was then quenched by perfusion with iodide. After achieving basal fluorescence, iodide was replaced with nitrate to determine the iodide efflux. The fluorescence in D555E‐transfected cells significantly increased compared to that in NBCe1‐transfected cells. These data indicate that the chloride conductance associated with D555E is most likely to be intrinsic to the mutant transporter.