Positive allosteric modulators of norepinephrine transporter

Solute carriers (SLCs) are proteins that transport essential molecules across cellular membranes. Hence, they play a crucial role in maintaining homeostasis in the body. Mutations in SLC genes, or in genes that regulate their expression and delivery to the cell surface, have been linked to various human diseases. In most cases, these mutations impair the transport function of the protein, and this loss of activity causes the disease symptoms. The current project focuses on a promising therapeutic approach: positive allosteric modulation for SLC activation. An allosteric modulator of a solute carrier is a molecule that binds at a location that differs from where the carrier normally binds its substrate. A positive allosteric modulator increases this activity, making the protein work more efficiently. While several allosteric binding sites were identified on proteins in the SLC6 family, all known modulators discovered so far decrease transport activity, i.e., they work as brakes rather than accelerators. Recently, butyrate was identified as a positive allosteric modulator of the serotonin transporter. Our preliminary experiments show that butyrate and a related compound, 4-hydroxybutyrate, also activate norepinephrine transporter (NET). This discovery demonstrates that NET has a binding site for positive allosteric modulators that can be targeted by organic molecules. Focusing on NET has important medical relevance because several clinical syndromes result from reduced NET activity. This project will investigate the mechanism by which butyrate enhances the transport of molecules through NET. To this end, we will use an electrophysiology technique called patch clamping, which allows monitoring NET function in real time. Patch clamp recordings will help to understand how butyrate influences the different steps of the transport process of NET. We will test a series of compounds with chemical structures similar to butyrate to identify high-affinity activators of NET. For this we will establish a high-throughput screen that allows testing a large number of compounds. The insights gained from this work will be valuable beyond NET. The principles we discover about how positive allosteric modulators work should apply to other members of the solute carrier family. Similarly, the screening assay we will develop can be adapted to search for activators of other transporters whose malfunction causes disease.