Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

Abstract

Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.NMDA receptors | ctenophores | crystal structures | evolution I n the nervous system and neuromuscular junction of many animal species, the amino acid L-glutamate acts as an excitatory neurotransmitter. The molecular organization of glutamate receptor ion channel (iGluR) subunits into an amino terminal domain (ATD), and a ligand binding domain (LBD) bisected by insertion of a pore loop ion channel generates a unique structural signature, distinct from that for other neurotransmitter receptors, that is easily identified by sequence analysis. Using this approach, hundreds of iGluR homologs are emerging from genome sequencing projects (1-5). Virtually all of these are glutamate receptors in name only; their functional properties, physiological function, and the ligands they bind have yet to be determined. Recent large-scale sequencing projects, which place ctenophores as candidates for the earliest metazoan lineage, reveal that iGluR homologs are abundantly represented in the genomes of the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, suggesting that glutamate was selected to act as a neurotransmitter very early in evolution (4, 5). The muscle cells of P. bachei respond to application of glutamate with action potential generation and both species have neural networks and exhibit complex predatory behaviors that might also be generated by iGluR activity (4, 5). However, as for most species studied in sequencing projects, ctenophore iGluRs have yet to be characterized.By contrast to our primitive state of knowledge for iGluRs recently discovered by genome sequencing projects, the iGluRs of vertebrate species have been extensively characterized, and based on their ligand binding properties, amino acid sequences...

Molecular lock regulates binding of glycine to a primitive NMDA receptor

“…ML032222a from the comb jelly M. leidyi forms a homomeric glycineactivated ion channel with rapid onset but extremely slow recovery from desensitization (14). We hypothesized that the unusually slow recovery from desensitization for ML032222a, which lasts for several minutes, occurs because the glycine bound LBD is trapped in a closed cleft conformation by a lock at the entrance to the binding site ( Fig.…”

“…The S1S2 LBD of ML032222a expressed as a soluble protein binds glycine with an affinity of 2.3 nM and is resistant to proteolysis by trypsin unless it is dialyzed in the unfolded state to remove endogenous glycine that remains bound during purification (14). Proteolysis protection assays revealed that similar to the WT protein, unfolding and dialysis in 4 M guanadinium was also required to prepare apo protein for the R703K mutant, whereas by contrast, for the E423S mutant, apo protein could be prepared by exhaustive dialysis without the need for unfolding ( Fig.…”

“…Multiple glycine-activated iGluR subunits have recently been discovered by genome sequencing of ctenophore species (24), including the comb jelly M. leidyi (14,25). A special feature of these subunits is an interdomain salt bridge that has not been found in iGluR subunits encoded in the genomes of a wide range of other organisms, including mammals.…”

“…Synthetic genes with codon optimization for expression in Escherichia coli for the ML032222a LBD S1S2 construct with point mutations were created by overlap PCR, expressed as soluble proteins, purified to homogeneity using metal affinity and ion exchange chromatography, and crystallized as described previously (12,14). X-ray diffraction data collected at Southeast Regional Collaborative Access Team (SER-CAT) Advanced Photon Source (APS) was used to solve structures for the glycine bound complexes of the R703K and E423S mutant LBDs by Fourier synthesis, using the WT structure with mutant side chains truncated to CB atoms, and with alternate conformations, ligand atoms, and solvent removed from the starting model.…”

“…We recently reported the discovery of glycine-activated iGluRs from the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, candidates for earliest lineage metazoans, for which ML032222a and PbGluR3 glycine complex crystal structures reveal a salt bridge at the perimeter of the ligand binding cleft (14). This salt bridge links the upper and lower lobes of the LBD in the closed cleft glycinebound conformation.…”