ORGANIC ANION TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 61

studies. 57 − 59 In this mechanism the transporter switches between two alternating
conformations, inward facing ( C i ) and outward facing ( C o ), thus allowing transloca-
tion of substrate across the membrane. Recently, a more detailed mechanism of the
conformational change has been proposed based on the crystal structure of a bacte-
rial antiporter, GlpT. 53 According to this mechanism (Figure 4.7), substrate binding
within the transporter binding site reduces the energy barrier between the inward- and
outward-facing conformations ( C i and C o ) and thus facilitates a transition between
them, involving a swiveling movement of the two six-TMD halves of the transporter
relative to each other (“rocker-switch” mechanism). Analysis of the density map of
another bacterial transporter, OxlT, suggests the existence of a third, “closed” confor-
mation of the transporter ( C c ) with the substrate-binding site isolated from both cyto-
plasm and periplasm, as an intermediate state in the transition between the C i and C o
conformations. 51
Importantly, in anion-transporting MFS proteins, including OATs, the binding
site supposedly comprises (is formed by) two positively charged amino acid residues.
These two key residues are, for example, Arg45 (located in the -helix H1) and Arg269

(H7) in GlpT, Arg46 (H1) and Arg275 (H7) in UhpT (sugar phosphate transporter
of E. coli ), Arg272 (H8) and Lys355 (H11) in OxlT, and Lys370 (H8) and Arg454
(H11) in rOAT3. 44 , 49 , 50 , 52 , 60 Based on the structure of GlpT, the two key positively
charged residues in the substrate-binding site were suggested to form hydrogen bonds
simultaneously with a negatively charged substrate ion. 52 , 53 The formation of such a
“bridge” complex (e.g., Arg45

α

Arg269 in GlpT) upon binding a substrate ion
to a transporter in the conformation open to one side of the membrane was suggested
as a mechanism to pull the N- and C-terminal domains closer to each other and make
further tilting easier (Figure 4.8).

··· P i ···

4.5. ANIMAL MODELS

As noted earlier, the evidence accumulated indicates that OAT1 and OAT3 manifest
functional properties and anatomical localization consistent with a critical role in the
basolateral uptake step of renal secretion of organic anions by the classical pathway.
However, most of this evidence has derived from in vitro studies, so that the actual in
vivo function of OATs, in the context of the whole kidney and/or the entire organism,
has remained uncertain. Recently, mice containing null alleles for OAT1 or OAT3
were generated using homologous recombination. Studies of OA transport in these
knockout mice have begun to define the role of OAT1 and OAT3 in vivo 61 , 62 and
their potential role in nephrotoxicity and drug-drug interactions (Figure 4.9). Some
of these results are discussed below.

4.5.1. Analysis of Transport Ex Vivo

Although OAT1 and OAT3 knockout mice are viable, fertile, and appear grossly
normal, they do manifest specific functional defects in renal organic anion handling