Ionotropic glutamate receptors (iGluRs) are a conserved family of ligand-gated ion channels present in both eukaryotes and prokaryotes. By regulating cation flow across the plasma membrane in response to binding of extracellular glutamate and related ligands, iGluRs represent an important signalling mechanism by which cells modify their internal physiology in response to external chemical signals.
iGluRs have originated by combination of protein domains originally encoded by distinct genes () 
. An extracellular amino-terminal domain (ATD) is involved in assembly of iGluR subunits into heteromeric complexes 
. This precedes the ligand-binding domain (LBD), whose two half-domains (S1 and S2) form a “Venus flytrap” structure that closes around glutamate and related agonists 
. Separating S1 and S2 in the primary structure is the ion channel pore, formed by two transmembrane segments and a re-entrant pore loop 
. S2 is followed by a third transmembrane domain of unknown function and a cytosolic carboxy-terminal tail.
A broad phylogenetic survey of iGluR and IR genes.
Animal iGluRs have been best characterised for their essential roles in synaptic transmission as receptors for the excitatory neurotransmitter glutamate 
. Three pharmacologically and molecularly distinct subfamilies exist, named after their main agonist: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). AMPA receptors mediate the vast majority of fast excitatory synaptic transmission in the vertebrate brain, while Kainate receptors have a subtler modulatory role in this process. NMDA receptors require two agonists for activation, glutamate and glycine, and function in synaptic and neuronal plasticity. Representatives of these iGluR subfamilies have been identified across vertebrates 
, as well as invertebrates, such as the fruit fly Drosophila melanogaster
, the nematode worm Caenorhabditis elegans
and the sea slug Aplysia californica 
While most iGluRs have exquisitely tuned synaptic functions, identification of iGluR-related genes in prokaryotic and plant genomes provided initial indication of more diverse roles for this class of ion channel. A bacterial glutamate receptor, GluR0, was first characterised in the cyanobacterium, Synechocystis PCC6803 
. GluR0 conducts ions in response to binding of glutamate and other amino acids in vitro
, suggesting a potential function in extracellular amino acid sensing in vivo
. The flowering plant Arabidopsis thaliana
has 20 iGluR-related genes, named GLRs 
. Genetic analysis of one receptor, GLR3.3, has implicated it in mediating external amino acid-stimulated calcium increases in roots 
We recently described a family of iGluR-related proteins in D. melanogaster
, named the Ionotropic Receptors (IRs) 
. Several lines of evidence demonstrated that the IRs define a new family of olfactory receptors. First, the IR LBDs are highly divergent and lack one or more residues that directly contact the glutamate ligand in iGluRs. Second, several IRs are expressed in sensory neurons in the principal D. melanogaster
olfactory organ, the antenna, that do not express members of the other D. melanogaster
chemosensory receptor families, the Odorant Receptors (ORs) and Gustatory Receptors (GRs) 
. Third, IR proteins localise to the ciliated endings of these sensory neurons and not to synapses 
. Finally, mis-expression of an IR in an ectopic neuron is sufficient to confer novel odour-evoked neuronal responses, providing direct genetic evidence for a role in odour sensing 
The identification of the IRs as a novel family of olfactory receptors in D. melanogaster provides a potential link between the well-characterised signalling activity of iGluRs in glutamate neurotransmitter-evoked neuronal depolarisation and a potentially more ancient function of this family in environmental chemosensation. In this work, we have combined comparative genomics, molecular evolutionary analysis and expression studies to examine the evolution of the IRs. Four principal issues are addressed: first, when did olfactory IRs first appear? Are they a recent acquisition as environmental chemosensors in D. melanogaster, or do they have earlier origins in insect or deeper animal lineages? Second, what is the most recent common ancestor of IR genes? Do they derive from AMPA, Kainate or NMDA receptors, or do they represent a distinct subfamily that evolved from the ancestral animal iGluR? Third, what mechanisms underlie the expansion and diversification of this multigene family? Finally, do IRs function only as olfactory receptors or are they also involved in other sensory modalities? Through answers to these questions, we sought insights into IR evolution in the context of the origins of iGluRs, the appearance and evolution of other chemosensory receptor repertoires and the changing selective pressures during animal diversification and exploitation of new ecological niches.