Solute Carrier (SLC) transporters are vital integral membrane proteins that act as a gateway to the intracellular compartments of our cells for various substrates like endogenous metabolites, nutrients from our diet, amino acids, xenobiotics and more. Moreover, they allow the movement of these important ligands across tissues and into appropriate organs to power the human body. With over 400 transporters known in the human genome and subdivided into 70 distinct families, these transporters form a complex network to maintain the body homeostasis for a wide range of substrates. Divergence of evolutionary conserved gene sequences that encode SLC transporters and alterations in gene expression lead to countless diseases and disorders, both common and rare, due to the disrupted homeostasis (e.g. metabolic disorders, neurologic disorders, immunologic diseases and more). While the clinical relevance is apparent in the field, about 30% of SLC transporters remain functionally uncharacterized, also known as orphan transporters, without an identified substrate. This presents a challenge in the quest to fully understand the complete physiological and pathological process in these diseases. Furthermore, it slows the field from advancing therapies in an effective manner that is safe and personalized. For many of these transporters, there is a huge need to further characterize or study certain components to better understand their function, like its native structure or the impact variation has on function.
This dissertation aims at tackling some of these challenges in three distinct SLC transporter families: SLC6, SLC7 and SLC22. We begin chapter 1 by providing the physiological significance of each transporter family in the human body and an overview of current knowledge of its function and substrate selectivity for specific transporter family members (SLC22A15, SLC22A16, SLC7A5, and SLC6A1). This provides sufficient context to then dive deep into a particular problem the field is facing. All of these transporters are also found primarily in brain tissues and several brain cell types. This presents a unique set of challenges and novelty, like guiding therapies in the long-term that can be delivered into the brain or designing experiments that recapitulates an accurate physiological environment.
The second chapter of this dissertation focuses on the SLC22 family, specifically the functional characterization of the orphan transporter SLC22A15. Since its discovery in fruit flies, this transporter had never been fully characterized for its substrate(s) and much less its physiological significance in humans. We sought out to characterize the biological role of SLC22A15, including its substrate selectivity, transport mechanism, tissue distribution, potential inhibitors and research any clinically relevant genetic association studies. Using a multi-faceted approach of computational and experimental methods, we were able to fully examine the role SLC22A15 plays as a member of the SLC22 family. Beginning with a large metabolomic study, we identified potential substrates for SLC22A15, including ergothioneine, carnosine and carnitine amongst others. Using a series of uptake and transport assays, we were able to confirm that SLC22A15 preferentially zwitterions, more specifically the antioxidants ergothionine and carnosine, carnitine and to a lesser affinity for cations. Additionally, using uptake assays, we saw that SLC22A15 functions in a sodium-dependent manner, provding clarity on its transport mechanism. Phylogenetic analysis and comparative structure modeling demonstrated that SLC22A15 is most closely related to zwitterion family members SLC22A4, SLC22A5, and SLC22A16, further confirming our experimental results that SLC22A15 is a zwitterion transporter. Doing a deep literature search on its identified substrates, we also concluded SLC22A15 significance in its coordinated transport of ergothioneine specifically in the brain where both substrate and transporter are found in high levels.
The third chapter focuses on the SLC7 family of L-type amino acid transporters or LATs. The LAT-1 transporter in specific, has been a popular drug target given its high mRNA expression in the blood-brain barrier and placenta, amongst others. This transporter has well characterized large neutral amino acid substrates like phenylalanine and tyrosine and drugs like L-DOPA and gabapentin. Additionally, previous studies have shown LAT-1 clinical relevance in neurologic disorders such as epilepsy and autism spectrum disorder (ASD), as well as several cancer types. In the past, LAT-1 has been exploited to treat cancer by inhibiting LAT-1 with experimentally synthesized compounds, and today there are two compounds being evaluated in clinical trials for its effective use in treating specific cancers. While this is exciting news, the field could benefit from studying LAT-1 biophysical properties to better guide therapies in the future. The goal of this chapter was to better understand LAT-1 inhibitor interactions, which would help provide a framework for developing future LAT-1 drug targets. Our approach was to combine metainference simulation and computational modeling tools with experimental testing to characterize LAT-1 inhibitor interactions. Our predictions and computational analyses showed that amino acids positions S66, G67, F252, G255, Y259, W405 in LAT-1 are critical residues for inhibitor binding and druggable sub-pockets in the outward-occluded conformation. We synthesized the top scoring predictive analogs of several amino acids that were thought to interact with these residues and tested them in HEK293 cells overexpressing LAT-1. We found 4 novel compounds that successfully inhibited LAT-1 transport including ‘KH13’ (IC50 = 11.8 µM), ‘EN14’ (IC50 = 16 µM), ‘EN2’ (IC50 = 63.3 µM), and ‘KH1’ (IC50 = 44.4 µM). Ultimately, this study provided an effective experimental basis for LAT-1 inhibitor discovery and possibly for other SLC transporters as well.
The fourth chapter aims at studying a large set of variants in the GAT-1 (encoded by SLC6A1) transporter of the SLC6 transporter family. GAT-1 is the main transporter for γ-aminobutyric acid (GABA) in the brain, an inhibitory neurotransmitter that communicates with postsynaptic GABA receptors. In the past decade or more, focus and urgency has been applied to finding a therapy or solution to the clinical phenotypes associated with missense mutations in GAT-1. This includes a wide range of pediatric onset epileptic seizures types, ASD, schizophrenia, and other neurodevelopmental delay or disorders. This is due to the fact that SLC6A1 is a single gene disorder, making missense mutations in one allele quite lethal. At the same time, this makes SLC6A1 a promising target for novel therapeutics such as gene therapy. However, before designing therapies, the field has yet to fully understand the major trend or impact on GAT-1 function that these mutations cause. This study used high throughput experimental methods to study 213 missense variants within the GAT-1 transporter and another 86 variants using high content imaging. We tested mutated GAT-1 in-vitro for GABA uptake and found that 100 variants exhibited severe loss-of-function effects (<17.9% of wildtype GAT-1 function), 25 variants loss-of function (>7.9% and <50.8%), 3 variants showed some evidence of gain-of-function effects (>147.5%) and 85 variants were comparable to wildtype GAT-1 activity (>50.8% and <147.5%). Further data analysis demonstrated that all observed disorders (e.g. seizures, developmental delay, ASD, and schizophrenia) in SLC6A1 de novo missense variants led to an overall loss-of-function mechanism. This was the largest functional characterization study of SLC6A1 missense variants and led to the suggested reclassification of about 77% of variants in our set of 213 that were classified as variants of unknown significance to either low or typical function. Next, we set out to visualize how these variants contribute to loss-of-function through high content imaging experiments by tagging the mutated GAT-1 transporters with GFP and seeing whether the protein traffics to the membrane properly. We found that variants with typical uptake were properly trafficking to the plasma membrane as expected, and that those causing low uptake of GABA did not traffic to the plasma membrane. A small subset of these variants was detected on the plasma membrane, but showed loss-of-function in uptake studies. These variants were clustered around the GABA binding pocket of GAT-1, explaining the third contradictory result. Using the recently solved cryo-EM GAT-1 structure, we were able to map out these variants on the 3D representation and using our results from the high content imaging experiments, inform the field on variant impact trends. Additionally, machine learning algorithms were used to analyze and select which variant impact prediction model best fit our results. The prediction tool ClinPred was the best predictor and we applied the tool to predict the GABA uptake for every amino acids residue in GAT-1 for future use. Collectively, this study informs the field on how variation impacts GAT-1 globally (loss-of-function) and that SLC6A1 is vulnerable to missense variation. Our results help guide the design of future therapies with a goal of restoring GAT-1 function in clinical phenotypes.
In summary, this dissertation led to the discovery of multiple components of very distinct SLC transporters in the brain. For SLC22A15, we discovered a new function and novel substrates. Our LAT-1 studies led to a framework of inhibitor discovery for future therapies targeting different cancer types. The largest functional genomics study for SLC6A1 revealed the major functional mechanism for 213 variants and has provided critical insight into how these variants contribute to clinical phenotypes in the CNS. Additionally, we identified the best predictive tool, ClinPred, using our data for future studies to implement. Collectively, these studies show the significance of uncovering more biological roles and impact of SLC transporters as they relate to diverse pharmacological targets and diseases causing genes.