Calcium signalling is fundamental for all eukaryotic cells. Its existence relies on the homeostatic maintenance of sub-micromolar levels of cytosolic calcium ([Ca2+]cyt). Abnormal perturbation of this basal level triggers a number of pathologies, from cancer to neurodegeneration and apoptosis. This proposal will focus on the provocative hypothesis that vesicular traffic and protein targeting by plant CORNICHON-homologue (CNIH) proteins have a direct regulatory role in [Ca2+]cyt homeostasis and signalling. My group was pioneer in showing that GLUTAMATE RECEPTOR-like (GLR) proteins are Ca2+ permeable ion channels in plants. CORNICHON proteins are ER cargo adaptors mediating the recruitment of integral membrane proteins into COPII vesicles. Here we present evidence that pairs of CNIHs are a necessary condition for the selective targeting of GLRs to specific endomembrane compartments, resulting in their differential localization to different Ca2+ stores. These results made us hypothesize that CNIHs themselves have a feed-back role in Ca2+ homeostasis by controlling the quantity and types of channels that are targeted to these stores. This hypothesis was further substantiated by our finding that the interaction between GLRs and CNIHs gate substantial ion currents in the absence of a ligand. We will test this hypothesis by a combination of genetics, quantitative Ca2+ imaging, mathematical modelling, electrophysiology and protein structural analysis, focusing on three specific aims. (1) We will manipulate CNIH action by over-expression, by changing molecular determinants of cargo sorting and domain swaps within the 5 CNIHs expressed in Arabidopsis. This will allow us to re-address or retain specific GLRs to different subcellular locations. We predict this will produce growth phenotypes by crossing [Ca2+]cyt homeostasis boundaries, which will inform us of the functional hierarchy of the trafficking mechanisms affected. (2) We will develop mathematical models to simulate the relevance of each sub-cellular location to [Ca2+]cyt. We will calibrate these models by screening a vast array of multiple, combined mutations in the GLR/CNIH families, and quantify their Ca2+ choreography changes and GLR localization. This approach will result in phenotypes that will reveal hierarchical contributions of each GLR/location set. Finally (3) we will study the physical interaction of CNIHs and GLRs by electrophysiology after heterologous expression in mammalian cells and by Cryo-EM. Results should enable us to establish a novel model of [Ca2+]cyt regulation based on vesicular trafficking mediated by CNIHs. We argue that this mechanism may be more visible and relevant in plants because they lack all the molecular machinery that animal cells evolved for coordinating small ligand operated Ca2+ stores (such as IP3 and ryanodine receptors, cyclases and phosphodiesterases). However, similar functional interactions between these two classes of proteins exist in yeast and animal cells, and thus we posit they may have an important role in animal cell physiology and pathology, thus potentially challenging the current paradigm of [Ca2+]cyt regulation in eukaryotic cells.