Developing Novel Protein-based GABA Sensor Modules Using PLP-dependent GabR Regulators

The proposed development of novel protein-based GABA sensor modules using PLP-dependent GabR regulators aims to facilitate the understanding of GABAergic neurotransmission. GabR, a bacterial transcription regulator, shows remarkable specificity for GABA through a reversible covalent bond with PLP, triggering conformational changes that produce (change of) fluorescence signals. In this proposal, we plan to create “genetically encoded” and chemically labeled “semisynthetic” fluorescent biosensors using GabR to detect real-time changes in GABA concentrations. GABA, a key inhibitory neurotransmitter in the central nervous system (CNS), is implicated in various mental health issues, including Depression, Schizophrenia, ADHD and Autism. Our effort first focuses on enhancing the stability and sensitivity of GabR-based biosensors by leveraging the intrinsic fluorophore in PLP and employing FRET signals from excitation of PLP. In addition, we also aim to optimize chemically labeled “semisynthetic” sensors with improved spectral tuning and kinetics for deep tissue imaging and real-time GABA sensing studies. These novel GABA sensors will be valuable tools for investigating GABAergic neurotransmission in live cells, brain slices, and in vitro assays, with potential applications in drug discovery and in vivo neurological research. Our work will significantly improve the understanding and monitoring of dynamic GABA levels in neurological systems, opening doors for further studies on mental health conditions and neuropharmacology. As an important part an R15(AREA) grant, educational and outreach efforts are designed in a cohesive structure to provide interdisciplinary trainings for graduate, undergraduate and high school students.