RFC-1 gene expression regulates folate absorption in mouse small intestine

Abstract

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-CH3-H4folate 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 μM) 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 μM) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l,L-5-CH3-H4folate 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 antimouse 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-[3H]-5-CH3-H4folate in a concentration-dependent manner. These results strongly suggest that pH- dependent folate absorption in this tissue is regulated by RFC-1 gene expression.