Structural and molecular determinants of glutamate transporter allosteric modulators.

Excitatory amino acid transporters (EAATs) are critical regulators of synaptic glutamate levels in the central nervous system. Dysregulated central nervous system glutamatergic homeostasis is implicated in many neurological diseases, highlighting the key role of EAATs in neurological health. We previously identified a library of small compounds that function as either positive allosteric modulators (PAMs) or negative allosteric modulators of EAATs, with diverse selectivity for subtypes EAAT1, EAAT2, and EAAT3, including astrocytic EAAT1 and EAAT2 and neuronal EAAT3. In this work, we characterized compounds from our library using molecular modeling, mutagenesis, and pharmacologic approaches. We focused on 3 representative compounds: NA-014, an EAAT2-selective PAM, DA-038, an EAAT1-3 PAM, and NA-010, an EAAT2-selective negative allosteric modulators. Binding studies demonstrated that these compounds do not interact with the orthosteric glutamate-binding site, confirming an allosteric action. Docking studies suggested several potential binding poses of NA-014 between the scaffold and transport domains of EAAT2, which we then studied with mutagenesis approaches. We identified potential binding sites of representative compounds in transmembrane domains 1, 5, 8, and hairpin 2 and demonstrated that these are necessary for their activity. Ten key amino acid residues within a subdomain of EAAT2 substituted into EAAT1 conferred EAAT2-selective PAM activity, demonstrating these residues are required and sufficient to enable selective PAM function. Collectively, these studies identified crucial subdomains and key amino acids linked to PAM activity, advancing our understanding of how to modulate EAAT activity. This knowledge can be integrated into future studies to develop EAAT allosteric modulators for neurological disorders. SIGNIFICANCE STATEMENT: We identified modulators of glutamate transporters, key regulators of central nervous system excitability and neuronal health. Using molecular modeling, mutagenesis, and pharmacology, we mapped their allosteric binding sites and identified ten residues that confer selective transport enhancement. This mechanism of transporter activation may guide development of therapies for disorders involving glutamatergic dysregulation, including stroke, neuropathic pain, and substance use disorders.