Structural Variation Governs Substrate Specificity for Organic Anion Transporter (OAT) Homologs

Abstract

Organic anion transporters (OATs, SLC22) interact with a remarkably diverse array of endogenous and exogenous organic anions. However, little is known about the structural features that determine their substrate selectivity. We examined the substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison with the more broadly expressed transporter, Oat1 (first identified as NKT). In analyzing interactions of both transporters with over 40 structurally diverse organic anions, we find a correlation between organic anion potency (pK i ) and hydrophobicity (logP) suggesting a hydrophobicity-driven association with transporter-binding sites, which appears particularly prominent for Oat6. On the other hand, organic anion binding selectivity between Oat6 and Oat1 is influenced by the anion mass and net charge. Smaller mono-anions manifest greater potency for Oat6 and di-anions for Oat1. Comparative molecular field analysis confirms these mechanistic insights and provides a model for predicting new OAT substrates. By comparative molecular field analysis, both hydrophobic and charged interactions contribute to Oat1 binding, although it is predominantly the former that contributes to Oat6 binding. Together, the data suggest that, although the three-dimensional structures of these two transporters may be very similar, the binding pockets exhibit crucial differences. Furthermore, for six radiolabeled substrates, we assessed transport efficacy (V max ) for Oat6 and Oat1. Binding potency and transport efficacy had little correlation, suggesting that different molecular interactions are involved in substrate binding to the transporter and translocation across the membrane. Substrate specificity for a particular transporter may enable design of drugs for targeting to specific tissues (e.g. olfactory mucosa). We also discuss how these data suggest a possible mechanism for remote sensing between OATs in different tissue compartments (e.g. kidney, olfactory mucosa) via organic anions.

Interaction of Organic Cations with Organic Anion Transporters

“…Oat6 did not appear to have the same degree or range of affinity for these compounds as did the other two transporters, although its relatively high affinity for nicotine is notable. These results support the view that although Oat1 and -3 share close structural homology and often overlapping substrate specificities, there are distinct differences in their binding sites (16,17). Furthermore, Oat6, although a close phylogenetic relation of Oat1, has been observed to have charge and hydrophobicity preferences distinct from those of Oat1 (16).…”

“…However, among the cations that bound to the Oats, only cimetidine and, to a lesser degree, 1-MPP were actually transported. This supports previous observations that substrate binding to the Oats and translocation across the membrane are two distinct processes (16), with the former being dependent on the physicochemical characteristics of the substrate and the latter on the molecular features of the substrate, which lead to transporter conformational changes. Finally, it seems worth considering in more detail whether other SLC22 transporters, including recently identified family members (11), might have the ability to bind and/or transport cations as well as anions.…”

“…Inhibition data were curve-fitted using nonlinear regression in Prism software 5.0 (GraphPad Inc., San Diego, CA) to calculate the K i Ϯ S.E. as before (16). K m of cations was determined by measuring uptake in the presence of 3-4 cation concentrations, followed by curve-fitting using nonlinear regression in Prism software, version 5.0.…”

“…We noted that a number of cationic compounds manifested significant binding affinity for Oat1 and -3 and, to a lesser degree, for Oat6 in the oocyte binding assays. To determine whether this was also true at the level of whole tissues, we examined the interaction of cationic compounds with transport of fluorescent organic anion substrates in cultures of kidney organ derived from Oat knock-out mice (16,17). Organ culture represents a living organ system where the Oat is in interaction with the cellular membrane and other transporters.…”

Protein Based Drug Discovery

“…However, little is known about the structural features that determine their substrate selectivity. The substrate binding preferences and transport function of olfactory organic anion transporter, Oat6, in comparison with the more broadly expressed transporter, Oat1 [65]. Long-term exposure to antivirals is associated with serious cellular toxicity to the kidney and other tissues.…”