MAMMALIAN OLIGOPEPTIDE TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 126

steroid hormones (progesterone and norethisterone at 3, 10, and 30 mM) for 24 hours
resulted in a significant decrease in the apical-to-basal transport of cephalexin in
Caco-2 cell monolayers. 156
6.4.7. Single-Nucleotide Polymorphisms
Single-nucleotide polymorphisms (SNPs) of transporter genes have also been investi-
gated as possible factors influencing interindividual variations in PK and PD profiles.
Coding region SNPs (cSNPs) of transporter genes may induce amino acid mutations,
resulting in changes in transporter intrinsic activity by altering the protein's affinity
to substrates ( K m ) and/or translocation ability or capacity ( V max ). 157 Despite increas-
ing efforts to understand the molecular and functional characteristics of oligopeptide
transporters, little information is available on their respective genetic polymorphisms.
Zhang et al. reported nine nonsynonymous and four synonymous cSNPs in the PepT1
gene in a population of 44 persons of different ethnic backgrounds. 158 Plasmids were
constructed by site directed mutagenesis for each of the nine nonsynonymous variants
and transiently transfected into HeLa cells for functional characterization. Transport
kinetics were studied utilizing Gly-Sar as a model substrate; and PepT1 expression
was determined by Western blot and immunocytochemical analyses. Results demon-
strated that among the nine nonsynonymous SNPs, P586L resulted in significantly
reduced transport capacity ( V max ) and decreased PepT1 protein expression levels. 158
Following its initial cloning from a human cDNA kidney library, a single PepT2
transcript was identified and accepted as a consensus sequence. 18 Currently, five
nonsynonymous cSNPs (R57H, L350F, P409S, R509K, and M704L) have been iden-
tified (http://www.ncbi.nlm.nih.gov/SNP/). 159 However, the functional activities of
only two of these variants, R57H and P409S, have been characterized functionally
in transiently transfected HEK293 cells. 159 Uptake studies performed using these
transients and contrasted with wild-types revealed elimination of Gly-Sar uptake in
the presence of the R57H variant. Interestingly, the P409S SNP had little effect on
function since comparable activity was observed in the transient comparative to the
wild-type hPepT2. Western blot analysis and immunofluorescence in Xenopus oocytes
expressing the PepT2 R57H variant verified protein expression at the membrane sur-
face, indicating that the loss of transport activity was not due to changes in expression,
but rather, in it functional characteristics.
6.4.8. Splice Variants
The importance of splice variants in general cannot be underestimated, as they increase
protein diversity by allowing multiple, sometimes functionally distinct proteins to be
encoded by the same gene. However, elucidating the function of each splice variant
has been a tremendous task, especially given the focus on the parent isoform. Inui
and colleagues first identified a novel pH-sensing regulatory protein cofactor which
modulates the transport activity of hPepT1. 160 The cDNA was cloned from human
duodenum having 1704 bp that encoded a predicted protein of 208 amino acid residues.