ORGANIC ANION–TRANSPORTING POLYPEPTIDES - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 79

of the protein to the cell surface. Moreover, each cysteine residue was found to be
disulfide bridged. Given that the electrostatic potential of extracellular loop 5 is not
basic, 3 it is not predicted to have functional interactions with solutes, highlighting
the important functional role of this domain in membrane insertion. Defining the
quaternary structure of OATPs has received little attention despite the perception that
members of this family form homo- or heterooligomers. For instance, even under
reducing conditions, high-molecular-weight bands suggesting multimers are present
after SDS-PAGE analysis of OATP1A2 protein heterologously expressed in mam-
malian cells. 13 Cross-linking experiments with OATP2B1 showed that amino groups
between two OATP2B1 molecules would have to be minimally 12 A apart to be consis-
tent with the observed homo-cross-linking found in cells overexpressing the protein. 12
Attempts to determine whether mouse Oatp1a1 and Oatp1a4 heterodimerize in liver
using immunopreciptation failed to show direct association between the proteins. 14
5.2.3. Transport Mechanisms
It is generally considered that transport via OATPs occurs in a bidirectional fashion
dictated by the solute gradients across the membrane. The mechanisms underlying
solute transport by the OATPs have been investigated in some detail, especially in
the context of bile acid physiology. Attention has focused largely on understanding
driving forces, especially since early in vitro studies intriguingly found a lack of
stimulation in OATP transport activity by an inwardly directed sodium gradient. 15 − 17
typical of that which is found for bile acid uptake into hepatocytes. Studies with rat
Oatp1a1 and Oatp1a4 first demonstrated that solute uptake into cells was energized by
countertransport with either bicarbonate 18 or reduced glutathione (GSH). 19 , 20 The sto-
ichiometry for GSH/bile acid exchange for Oatp1 is 1 : 1. 19 However, Oatp1-mediated
GSH efflux was not dependent on obligate exchange with solute such as bile acids. 21
Interestingly, human OATP2B1 was found to possess pH-dependent transport proper-
ties that were solute selective. 17 , 22 Extracellular acidification promoted solute uptake,
a property of OATP2B1 that bears relevance to the environment in which the trans-
porter is expressed on the apical membrane of enterocytes. pH-stimulated transport by
OATP2B1 is in contrast to the insensitivity of rat Oatp1 activity to proton gradients. 23
A predominant bile acid efflux function for OATP1B3 in liver has been proposed. The
fascinating finding that bile acid transport by OATP1B3 and not OATP1B1 occurs
by a GSH cotransport mechanism raises the possibility that the functional transporter
affords protection to hepatocytes by limiting the accumulation of toxic intracellular
solutes. 24 A 2 : 1 GSH/bile acid cotransport stoichiometry for OATP1B3-mediated
transport was observed. OATP2A1, the prostaglandin transporter PGT, appears to
energize solute uptake by outward exchange with lactate. 25 Overall, it is becoming
evident that the various OATPs can be defined by different transport mechanisms.
Structural modeling of OATP1B3 and OATP2B1 has suggested that OATPs trans-
port solutes across membranes through a rocker-switch type of mechanism, 3 but the
location of solute-binding sites and molecular mechanisms of the transport process
are unclear. How the transport mechanisms relate with physiological roles of OATPs
remains poorly understood.