When Metabolism Became Messaging: The Stepwise Evolution of the GABAergic Signaling System

The GABAergic system is the principal inhibitory signaling machinery in bilaterian nervous systems, yet its evolutionary assembly remains unresolved. Here, we reconstruct the origin and diversification of the complete GABAergic toolkit-including biosynthetic and catabolic enzymes, transporters, and ionotropic and metabotropic receptors-across 89 proteomes, representing 53 metazoan species and 36 outgroup lineages using phylogenomics and gene tree-species tree reconciliation. Our analyses reveal that GABAergic signaling did not emerge as a unified synaptic system but assembled stepwise from pre-existing metabolic components. Core enzymes of the GABA shunt (GAD1 and ABAT) predate nervous systems and are present in basal metazoans, indicating an ancestral metabolic or paracrine role. The emergence of GABA signaling in Cnidaria coincides with the recruitment of plasma membrane transporters and metabotropic GABA-B receptors, supporting early modulatory functions. In contrast, key components required for fast synaptic inhibition-vesicular transporter VIAAT and ionotropic GABA-A receptors-appear only in bilateria, marking a major functional transition. The asymmetric distribution of GABA-B subunits further suggests an ancestral promiscuous signaling state prior to obligate heterodimerization. Together, these findings support a three-stage evolutionary model in which inhibitory neurotransmission arose through progressive co-option and specialization of ancient molecular modules over ~500-600 million years.