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Membrane transporters are integral proteins contributing to the cellular integrity of all tissue and cell types. The solute carrier superfamily represents the second largest family of membrane proteins, encoding for over 400 transport proteins that collectively, play a pivotal role in cellular homeostasis, including the transport of essential nutrients or ions and the removal of toxic by-products. Moreover, membrane transporters are major contributors to the pharmacokinetics of therapeutic drugs, and the tissue specific expression of uptake transporters can serve as initiating mechanisms that govern a drug’s pharmacodynamic properties (e.g., efficacy and toxicity). Here, we demonstrate that the sub-family of organic cation transporters are critical mediators initiating the debilitating toxicity profiles of chemotherapeutic agents, and that the targeting of these transporters can be exploited clinically to afford protection against injury in healthy tissues without compromising therapeutic benefits.

The SLC22 family of transmembrane transport proteins consists of at least 31 known members that transport organic anions (OATs), organic cations (OCTs) and zwitterions (OCTNs). Despite their interaction with many endogenous metabolites and signaling molecules, many of these transporters are referred to as “drug” transporters since they facilitate the movement of pharmaceutical drugs. However, their evolution and conservation as a group underscores key endogenous functions. Chapter 1 suggests a re-designation of SLC22 into nine subclades with four new, functional subclades arising out of the previously defined Oat subclade - referred to as Oat subgroups. These new Oat subgroups are: OATS1 (SLC22A6, SLC22A8, and SLC22A20), OATS2 (SLC22A7), OATS3 (SLC22A11, SLC22A12, and slc22a22), and OATS4 (SLC22A9, SLC22A10, SLC22A24, SLC22A25 plus the rodent expansion of slc22a19, and slc22a26-30). We also propose the reclassification of SLC22A16 into the preexisting OCTN subclade (SLC22A4, SLC22A5, and slc22a21) and the renaming of the Oct-related subclade, which now contains only SLC22A15, to the Octn-related subclade. Due to the functional similarities between the Drosophila melanogaster (fruit fly) hindgut and Malpighian tubules and the human intestines and kidneys, where most, if not all SLC22 family members can be localized, we are assessing the functionality of SLC22 transport proteins using flies as a model organism. Chapter 2 explores orthologous relationships between D. melanogaster transporters and their human counterparts. It also assesses fly transporters for their role in management of oxidative stress.

Members of the Solute Carrier (SLC) 22 family of transporters have been studied extensively due to their well-known role in drug excretion and disposition. However, numerous lines of evidence suggest that these transporters are likely to possess previously unrecognized roles in basal physiological processes. These studies were undertaken to better delineate the potential physiological importance of 2 organic anion transporters (OATs) expressed on the apical membrane of the apical membrane of renal tubule cells, SLC22A11 (OAT4) and SLC22A12 (URAT1), the impact of genetic variants of these two transporters, and to create a humanized mouse model suitable for an additional physiological and drug disposition studies of another SLC22 transporter, SLC22A1 (OCT1).Of the 9 OAT4 nonsynonymous variants studied, the L29P, R48Stop, and H469R variants all displayed negligible uptake of 3 canonical, endogenous substrates (estrone sulfate, ochratoxin A, and uric acid) likely due to the absence of plasma membrane protein localization. Extending this research to URAT1, 4 nonsynonymous variants, G65W, I131V, W277R, and R342H, as well as a C-terminal frameshift variant T542LysfX13, all displayed some reduction in uptake of uric acid, with the T542LysfX13 reduction likely due to the loss of a protein-binding motif and resultant plasma membrane localization. Collectively, the presence of these variants provides mechanistic reasons for variability in reabsorption of solutes from the urine in the human population, potentially affecting metabolite homeostasis as well as drug pharmacokinetics.Comparisons of the evolutionary, genetic, and molecular features of OAT4 and URAT1 demonstrated that both are under general purifying selection and that their evolution has diverged considerably within the mammalian lineage, with numerous species lacking clear orthologs for either transporter. Likewise, expression of OAT4 was also found in the epididymis, an embryonically related tissue to the kidney but involved in the maturation of spermatozoa. In addition, profiling of substrate preferences for these two transporters showed considerably divergent sets of interacting molecules, including the uptake of AMP and ADP by URAT1 and an apparent binding site size restriction difference. These results suggest that these transporters, while evolutionarily related, have diverged to accommodate specialized physiological roles in a subset of mammalian lineage.Finally, a mouse model expressing human OCT1 in the liver was created using the mouse albumin core promoter and distal enhancer to drive liver-specific expression. Absolute OCT1 transgene expression levels were 4 times higher in the transgenic line compared to the normal human liver and 20 times higher than any other mouse tissue. While expression was not restricted to the liver as anticipated, this model can aid in the assessment of drug substrates of OCT1 and provide a flexible model to evaluate the physiological repercussions of human OCT1 in an intact animal model.This work demonstrates that while these transporters are important from a drug development standpoint, they also may be involved in specialized physiological processes, which may be modulated by naturally occurring genetic variants. Additional studies are required to better understand the full impact of these transporters on human physiology and the resultant impact on human health.