Gamma-aminobutyric acid (GABA) is a potent neurotransmitter produced in the islet at levels as high as in the brain. While the function of GABA in the nervous system is well-understood, the description of the islet GABA system is clouded by dozens of antithetical reports describing differing secretion pathways and effector functions. It is now clear that GABA does not directly regulate beta cell mass, so GABA’s ultimate role in the islet remains unresolved. We recently described a new mechanism for GABA secretion from human islets that challenges the 30-year-old conceptual status quo that islet GABA secretion occurs via synaptic-like vesicles. Instead, beta cells release GABA directly from the cytosol via volume regulated anion channels (VRACs). Next, we showed that beta cells release GABA in regular pulses that provide periodic feedback to help synchronize hormone secretion. GABA is also metabolized in the beta cell through a pathway called the GABA-shunt, which can accelerate ATP production. We conducted pharmacological studies to manipulate GABA synthesis and catabolism, which profoundly impacted glucose-responsive insulin secretion. These results establish that GABA is important for normal islet function. From here, our Aims over the next five years are to (1) analyze the detailed mechanism of GABA efflux from human beta cells and (2) determine the overall role of GABA in glycemic control. Our approach implements two strains of Cre-Lox conditional knockout mice: beta cell-specific deletion of VRAC, the channel responsible for GABA release; and beta cell-specific deletion of GAD67, the enzyme responsible for GABA biosynthesis. The latter model represents the first example of an islet-specific GABA-null mouse. These models will be combined with technological innovations including GABA biosensor cells, islet-on-a-chip microfluidic devices, and optical probes for cytosolic Ca2+, membrane potential, and VRAC activity to dynamically measure islet GABA release and its functional effects. We will validate our conclusions in human islets including the use of live human pancreas organotypic slices. This research has relevance for human health. We previously found that GABA content and secretion are impaired in human islets from donors with type 1 and type 2 diabetes, suggesting GABA levels correlate with diabetes pathogenesis. Our proposed work will establish if there is a causal linkage between GABA and islet function. If successful in elucidating GABA mechanisms that affect islet hormone secretion, existing pharmaceuticals that modulate GABA systems can be proposed as novel intervention strategies to promote islet function in diabetes prevention or management. The immediate impact of this study will be to finally bring clarity to the role and mechanisms of the GABA system in islets. However, this research has broader impacts that extend to other neurotransmitters (VRAC is also permeable to glycine, glutamine, and taurine) and other cell type that utilize GABA including neurons, glial cells, enteroendocrine cells, and immune cells. Our team has extensive knowledge of human islet biology and unique technical expertise to succeed in this endeavor.