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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Departments of 1Internal Medicine, and 2Cellular and Molecular Physiology, and 3Surgery, Yale University, New Haven, Connecticut 06520
Submitted 29 August 2002 ; accepted in final form 16 June 2003
Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously
been identified in the basolateral membranes of rat colonic epithelial cells.
The present study demonstrates Cl-HCO3 exchange as the mechanism
for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in
the presence of a protease inhibitor cocktail from rat distal colon. Studies
of 36Cl uptake performed with BLMV prepared with different types of
protease inhibitors indicate that preventing the cleavage of the COOH-terminal
end of AE2 protein by serine-type proteases was responsible for the
demonstration of Cl-HCO3 exchange. In the absence of voltage
clamping, both outward OH gradient (pHout/pHin: 7.5/5.5)
and outward HCO3 gradient stimulated transient 36Cl
uptake accumulation. However, voltage clamping with K-ionophore, valinomycin,
almost completely (87%) inhibited the OH gradient-driven 36Cl
uptake, whereas HCO3 gradient-driven 36Cl uptake was
only partially inhibited (38%). Both electroneutral HCO3 and OH
gradient-driven 36Cl uptake were 1) completely inhibited
by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant
(Ki) of 
26.9 and 30.6 µM, respectively,
2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic
acid(NPPB), a Cl channel blocker, 3) saturated by increasing
extravesicular Cl concentration with a Km for Cl of
12.6 and 14.2 mM, respectively, and 4) present in both surface
and crypt cells. Intracellular pH (pHi) was also determined with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester
(BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted
in a DIDS-inhibitable increase in pHi both in
HCO3-buffered and in the nominally HCO3-free buffered
solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively).
We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange
is present in basolateral membranes and that, in the absence of
HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange
and regulate pHi across basolateral membranes of rat distal
colon.
crypt glands; superfusion; intracellular pH; membrane vesicles; 36Cl uptake; Cl-anion exchange
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