MAMMALIAN OLIGOPEPTIDE TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 117

transport process. 89 Furthermore, Fei et al. demonstrated that TMD7 to TMD9 play
a role in defining substrate affinity. 25 Doring et al. also determined that the first 59
amino acid residues of both PepT1 and PepT2 contribute significantly to substrate
affinity. 89 The functional significance of the C-terminal region of oligopeptide trans-
porters is unknown at this time; however, portions of the C-terminus may aid in trans-
porter trafficking, membrane insertion, and/or transporter regulation, as evidenced
by its inclusion in the functionally active rat pineal gland PepT1 (pgPepT1) splice
variant. 91
Studies using the substituted cysteine accessibility method (SCAM) have pro-
vided more detail concerning PepT structure. 85 , 92 TMD5, the most conserved region
across species, displays solvent accessibility along its entire length, lining the pu-
tative aqueous channel akin to related antiporters. 85 Similarly, TMD7 also exhibits
solvent accessibility. Preliminary molecular modeling, along with some experimental
evidence, has suggested that the extracellular end of TMD7 may shift following sub-
strate binding, providing the basis for channel opening and substrate translocation. 92
However, additional studies are required to clearly elucidate the molecular PepT
structure. Taken together, these findings suggest that the N-terminal TMDs form a
porelike structure, while TMD7 to TMD9 compose the substrate-binding pocket. 12

6.3.3. General Substrate Specificities

The three-dimensional structure of any of the POT proteins has not been elucidated. As
such, the structural requirements for molecular recognition have been based on uptake
or transport experiments, as well as on computational modeling. Several studies have
summarized the primary chemical moieties that a peptidomimetic molecule should
contain to function as a PepT substrate or inhibitor. 11 , 93 For years it was believed
that a peptide bond was essential for a molecule to have affinity for PepT, however,
nowadays it has been well established that this is not the case. 94 - 97 Furthermore, an
entire range of molecules, with varying functional moieties, are also transported by
PepT1 with affinity constants similar to those of some dipeptides. 98

Distinctively,

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substrates or inhibitors with high PepT1 affinity ( 0.5 mM) have been recognized as

molecules containing the following characteristics: (1) L-amino acids, (2) an acidic or
hydrophobic moiety at the C-terminus, (3) a weakly basic group in

-position at the
N-terminus, (4) a ketomethylene or acid amide bond, and in the case of a molecule
with a peptide bond, (5) to have a trans conformation. 99 Bailey et al. have corroborated
experimentally experimentally some of the structural characteristics already proposed
and identified some structural requirements necessary to increase PepT1 affinity for
a D-enantiomer over its corresponding L-enantiomer. 100 Furthermore, analysis of the
binding and transport characteristics of PepT1 have led to the development of several
molecular models attempting to establish a PepT-substrate template. 93 , 101 - 105
Comparatively, PepT1 is considered a low-affinity ( K m of 200 M to 10 mM), high-

capacity transport system, while PepT2 has a higher substrate affinity ( K m of 5 to 500

M) with a lower transport capacity. Interestingly, PepT2 affinity has been attributed
to the requirement of an -aminocarbonyl moiety for substrate recognition. 90

Moreover, the general structural requirements for PepT2 substrate or inhibitor

-or