pH modulation of the kinetics of rabbit jejunal, brush-border folate transport

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Abstract
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 μM) 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 μM). 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 μM;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 μM, 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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