Merging Signaling with Structure: Functions and Mechanisms of Plant Glutamate Receptor Ion Channels, and their implications for the evolution of aminoacid based ion-signaling systems - PROJECT SUMMARY Despite not having a nervous system, plants have exceptional capacities to communicate between cells and organs. Being sessile, this is vital for adaptation to animal herbivory (organ regeneration), extreme temperatures, pathogens at all levels, etc. Interestingly, plants have evolved an expanded family of glutamate receptors, proteins gated by glutamate to generate electric and calcium signals, which are well known for their crucial role in the human nervous system and many of its related pathologies. My lab has been leading the field of plant GLutamate Receptor (GLR) research. We pioneered their demonstration as ion channels involved in cell-cell communication, and, among other, further studied their evolution, roles in sexual reproduction, or functions of GLR-associated regulatory proteins. Under grant R01GM131043 we extended our scope to generate the first structural views of GLRs. Cryo-EM revealed a surprising conservation of structural mechanisms with their animal homologues (iGluRs) but also many differences, e.g. ligand promiscuity, non-canonical gating properties and different ionic selectivity. Globally these features revealed functions in electrical communication and ligand-gated Ca2+ store homeostasis for GLRs. Taking advantage of novel methodology that permits reasonable prediction from sequence and homologous structures, we are now re-iterating GLR function and their physiological roles by generating protein activity predictions by structural and molecular dynamics computational simulations. Specifically, we are targeting ligand specificity, structural topologies affecting gating and de-sensitization speeds, and ion selectivity. We posit these properties to be crucial for GLR-based physiology and will explore this strategy to break the barrier posed by genetic/functional redundancy resulting from the elevated gene copy number (20 in Arabidopsis, many more in other plants; 14 in humans). Our current efforts are focused in developing these approaches and establishing new protocols for validation of structural predictions from modeling. These will include validation of altered GLR properties by expression of combinations of mutated glutamate receptors and accessory proteins on heterologous systems. Modifications likely to generate either dominant-negative or -positive functional phenotypes will be knocked into our reference collection of GLR mutants and screened for function. We will target complementation of backgrounds with multiple mutations in diverse clades, and will screen for plausible functions of GLR roles, namely innate immunity, organ regeneration and abiotic stresses. Ion imaging and single-cell transcriptomics of promising lines will be further investigated for signaling and transcriptional pathways which are affected by GLR function. Given that plant GLRs evolved physiological functions out of topologies and motifs that are associated with disease when present in human glutamate receptors, notably in the “orphan” Glu-∂ sub-family, our results should also address intriguing medical questions related to these structural aspects.
