pH modulation of the kinetics of rabbit jejunal,brush-border folate transport |
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Authors: | Charles M Schron |
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Institution: | (1) Division of Digestive Diseases, Department of Internal Medicine, University of Cincinnati College of Medicine, 45267 Cincinnati, Ohio |
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Abstract: | Summary In jejunal brush-border membrane vesicles, an outwardly directed OH– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest.
76:2030–2033), suggesting carrier-mediated folate: OH– exchange (or phenomenologically indistinguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pHint/pHext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH 6.0. Since stepwise increments ininternal pH (4.5 7.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.35 6.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+
K
m
=12.8±1.2 M). Hill plots of these data suggest involvement of at least one H+ (OH–) at low pH (monovalent F– predominates) and at least 2 H+ (OH–) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F– and F–2 is electroneutral. Kinetic parameters for F– and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pHext5.8 (K
m
=1.33vs. 1.70 M;V
max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV
max, but does not affect theK
m
for carrier-mediated F transport. These data are consistent with similarK
i
'
s for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H+
K
m
(or OH– lC50) corresponding to pH 4.89. External pH effects theV
max, but not theK
m
for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH– or H+),rather than competitive binding that is mutually exclusive. |
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Keywords: | anion exchange intestinal transport vesicle transport |
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