# Detection of Calcium waves in roots of seedlings as a primary genetic screen for GLR and CNIH function

> **NIH NIH R01** · UNIV OF MARYLAND, COLLEGE PARK · 2020 · $71,425

## Abstract

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 ...

## Key facts

- **NIH application ID:** 10135545
- **Project number:** 3R01GM131043-01S1
- **Recipient organization:** UNIV OF MARYLAND, COLLEGE PARK
- **Principal Investigator:** Jose A Feijo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $71,425
- **Award type:** 3
- **Project period:** 2019-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10135545

## Citation

> US National Institutes of Health, RePORTER application 10135545, Detection of Calcium waves in roots of seedlings as a primary genetic screen for GLR and CNIH function (3R01GM131043-01S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10135545. Licensed CC0.

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