Molecular mechanism of the intestinal biotin transport process

Previous studies have characterized different aspects of the cellular/membr ane mechanism and regulation of the intestinal uptake process of the water- soluble vitamin biotin. Little, however, is known about the molecular mecha nisms of the uptake process. In this study, we have identified a cDNA from rat small intestine that appears to be involved in biotin transport. The op en reading frame of this cloned cDNA consisted of 1,905 bases and was ident ical to that identified for the vitamin transporter in placental tissue. Si gnificant heterogeneity, however, was found in the 5' untranslated region o f this clone, with three distinct variants (II, III, IV) being identified i n the small intestine; the placental variant (variant I), however was not p resent in the small gut. Variant II was found to be the predominant form ex pressed in the rat small and large intestines. Functional identity of the c loned intestinal cDNA was confirmed by stable expression in COS-7 cells, wh ich showed a four- to fivefold increase in biotin uptake in transfected COS -7 cells compared with controls. The induced biotin uptake in transfected C OS-7 cells was found to be 1) Na+ dependent, 2) saturable as a function of concentration with an apparent K-m of 8.77 mu M and a V-max of 779.7 pmol.m g protein(-1).3 min(-1), and 3) inhibited by unlabeled biotin and pantothen ic acid and their structural analogs. The distribution of complementary mRN A transcripts of the cloned cDNA along the vertical and longitudinal axes o f the intestinal tract was also determined Results of this study describe t he molecular characteristics of the intestinal biotin absorption process an d report the identification of a cDNA that encodes a Na+-dependent biotin u ptake carrier that appears to exist in the form of multiple variants.