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Exposing nature’s view of ligand recognition in ionotropic glutamate receptors

Periodic Reporting for period 1 - iGLURs - A NEW VIEW (Exposing nature’s view of ligand recognition in ionotropic glutamate receptors)

Reporting period: 2019-03-01 to 2020-08-31

"Ionotropic glutamate receptors (iGluRs) are the proteins in our brain that allow brain cells to communicate with each other so rapidly, enabling e.g. thoughts, behaviour, learning, and memory. Not surprisingly, their malfunction is implicated in numerous neurological disorders. Despite access to human and other animal genomes and of despite profound knowledge on certain aspects of iGluR function, we are still unable to look at DNA sequences and accurately predict iGluR function (or malfunction). This project aims to reach this level of knowledge. To do so, we aim to uncover how diverse the iGluR family is; how iGluRs evolved to be what they are now; and the chemical details of glutamate binding to iGluRs- in other words, ""Nature's view of ligand recognition in iGluRs"". This will be approached by techniques such as computational phylogenetics, electrophysiological experiments, and chemical biology manipulations of iGluRs. At the end, we hope to understand where iGluRs came from and how human - and all other - iGluRs function, uncovering a key aspect of nervous system evolution and laying bare the blueprint for medicinal chemistry targeting a vital protein in the brain."
Objective 1 - Establishing a broad and representative sample of iGluRs
Computational phylogenetics have resulted in a draft phylogenetic tree of the iGluR family that (1) recapitulates major relationships suggested by other researchers and (2) shows information from types of animals that were previously not studied. This has revealed some interesting patterns in iGluR evolution that we hope to pursue experimentally now. Several iGluR genes have been identified and synthesized, including iGluRs from comb jellies and sea anemones. Unfofrtunately, these are more difficult to work with in the lab than e.g. rat iGluRs, which we also use in the lab, but the ERC PhD student working on this has identified ways of boosting iGluR function in the lab, enabling the testing of these obscure iGluRs.
Objective 2 - Identifying the molecular changes that led to ligand recognition
This involves the forming and testing hypotheses that emerge from Objective 1. As objective 1 is ongoing, this has not been addressed yet. But the phylogenetics in Objective 1 are progressing, and already hypotheses are emerging that we will soon test functionally.
Objective 3 - Chemical interactions that determine ligand recognition
Substantial progress has been made on this front, starting with rat iGluRs. The PI and the ERC postdoc have established a system where we can incorporate non-cannonical amino acids into proteins (iGluRs). This has already enabled the postdoc to show that in rat NMDA receptors, a type of iGluR that is crucial for learning and memory, the chemical interactions that determine ligand recognition are different to what was previously thought. She is extending these preliminary results and also trying to combine them with Objectives 1 and 2 to establish broader reaching knowledge.
With others in the lab funded by other sources, we've been able to extend some of the work to examine the expression of iGluRs in model organisms with simplified nervous systems. An example is illustrated in the picture below.
So far, we have established techniques and made sure things are working, involving some teaching, learning, and optimization. However the above work in Objective 3 has already shown a surprising result, which isntead of confirming previously head assumptions, rather overturns them.