Functional analysis of ligand-gated chloride channels in a cnidarian sheds light on the evolution of inhibitory signalling
{psi}-aminobutyric acid (GABA) is the predominant inhibitory transmitter in the vertebrate nervous system. Fast inhibitory signalling is mediated by type A GABA receptors (GABAARs), which form pentameric ligand-gated ion channels. While GABA is also present in plants and prokaryotes, it is unknown when it was first used for fast neuronal transmission. Cnidaria represent a sister group to all Bilateria and possess a variety of putative GABAARs, none of which has been functionally characterized. In this study, we surveyed putative inhibitory ion channel receptors from the model cnidarians Nematostella and Hydra. Phylogenetic analysis revealed a surprising complexity of these receptors. The majority formed a cnidarian-specific radiation with some receptors forming a basal clade. We functionally analyzed seven putative Nematostella GABAARs of this radiation and found that none was activated by GABA or glycine, whereas three were activated by glutamate. Using site-directed mutagenesis, we identified a lysine residue in the canonical ligand-binding pocket that is important for activation by glutamate. Our results identified a group of inhibitory ion channel receptors in Cnidaria that uses glutamate as a ligand. Moreover, they suggest that inhibitory ion channel receptors in Cnidaria massively diversified, which may have been instrumental in the evolution of complex behaviors and sensory processing by the cnidarian nervous system. This work lays the foundation for understanding the diversity and evolution of inhibitory receptors in Cnidaria and the evolution of inhibitory signalling in animal nervous systems.