RFC-1 Gene Expression Regulates Folate Absorption in Mouse Small Intestine

Mediated folate compound transport inward in isolated luminal epithelial cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensitivity to the stilbene inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. pH dependence was manifested as higher maximum capacity ( V max ) for influx of l , L -5-CH 3 -H 4 folate at acidic pH compared with neutral or alkaline pH with no effect on saturability ( K m ). The pH-dependent component was relatively insensitive to inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and highly saturable ( K m or K i = 2 to 4 μ M ) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors. The non-pH-dependent component was highly sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and poorly and variably saturable ( K m or K i = 20 to >2000 μ M ) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l , L -5-CH 3 -H 4 folate influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was associated with an increase in RFC-1 gene expression in the form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane protein detected by folate-based affinity labeling and with anti-mouse RFC-1 peptide antibodies. The size of this protein was the same as that encoded by RFC-1 mRNA. The treatment of mature absorptive cells with either the affinity label or the anti-RFC-1 peptide antibodies inhibited influx of l , L -[ 3 H]-5-CH 3 -H 4 folate in a concentration-dependent manner. These results strongly suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.