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J. Biol. Chem., Vol. 277, Issue 13, 11004-11012, March 29, 2002
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From the The bumetanide-sensitive
Na+:K+:2Cl
Functional Properties of the Apical
Na+-K+-2Cl
Cotransporter
Isoforms*
,§¶,,
Molecular Physiology Unit, Instituto de
Investigaciones Biomédicas, Universidad Nacional Autónoma
de México and Instituto Nacional de Ciencias Médicas y
Nutrición Salvador Zubirán, Tlalpan 14000, Mexico City,
Mexico and the § Department of Cellular and Molecular
Physiology, Yale University Medical School,
New Haven, Connecticut 06520
cotransporter
(BSC1) is the major pathway for salt reabsorption in the apical
membrane of the mammalian thick ascending limb of Henle. Three isoforms
of the cotransporter, known as A, B, and F, exhibit axial expression along the thick ascending limb. We report here a functional comparison of the three isoforms from mouse kidney. When expressed in
Xenopus oocytes the mBSC1-A isoform showed higher capacity
of transport, with no difference in the amount of surface expression.
Kinetic characterization revealed divergent affinities for the three
cotransported ions. The observed EC50 values for
Na+, K+, and Cl were 5.0 ± 3.9, 0.96 ± 0.16, and 22.2 ± 4.8 mM for
mBSC1-A; 3.0 ± 0.6, 0.76 ± 0.07, and 11.6 ± 0.7 mM for mBSC1-B; and 20.6 ± 7.2, 1.54 ± 0.16, and 29.2 ± 2.1 mM for mBSC1-F, respectively. Bumetanide sensitivity was higher in mBSC1-B compared with the mBSC1-A
and mBSC1-F isoforms. All three transporters were partially inhibited by hypotonicity but to different extents. The cell
swelling-induced inhibition profile was mBSC1-F > mBSC1-B > mBSC1-A.
The function of the Na+:K+:2Cl
cotransporter was not affected by extracellular pH or by the addition
of metolazone, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid
(DIDS), or
R(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1-H-indenyl-5-yl)-oxy]acetic acid (DIOA) to the extracellular medium. In contrast, exposure of
oocytes to HgCl2 before the uptake period reduced the
activity of the cotransporter. The effect of HgCl2 was
dose-dependent, and mBSC1-A and mBSC1-B exhibited higher
affinity than mBSC1-F. Overall, the functional comparison of the murine
apical renal-specific Na+:K+:2Cl
cotransporter isoforms A, B, and F reveals important functional, pharmacological, and kinetic differences, with both physiological and
structural implications.
*
This work was supported in part by Research Grants 97629m
from the Mexican Council of Science and Technology (CONACYT) and 75197-553601 from the Howard Hughes Medical Institute (to G. G.) and
DK36803 from the National Institutes of Health (to S. C. H. and
G. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Molecular
Physiology Unit, Vasco de Quiroga 15, Tlalpan 14000, México City,
Mexico. Tel.: 525-513-3868; Fax: 525-655-0382; E-mail:
gamba@conacyt.mx.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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