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

Periodic Reporting for period 2 - iGLURs - A NEW VIEW (Exposing nature’s view of ligand recognition in ionotropic glutamate receptors)

Reporting period: 2020-09-01 to 2022-02-28

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 ionotropoc glutamate receptor (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 have begun pursuing experimentally now.

Several iGluR genes have been identified and synthesized, including iGluRs from comb jellies, sea anemones, and worms. These have been challenging to work with in our electrophysiological experiments, especially the genes from distantly (from rat and human) related comb jelly and sea anemone genes (detailed further in Part B, Major Achievements and Major Challenges). However, the ERC PhD student working on this has identified ways of boosting iGluR function in our experiments and she has now started accumulating data on the function of these different receptors, particularly in sea anemones and worms, and this is starting to lead to an hypothesis on changes in iGluR identity when the lineage from more complex animals (worms, flies, crustaceans, rats, humans; collectively "bilaterians") diverged from simpler animals (sea anemones, etc; collectively "cnidarians"). She is now (1) pursuing this work with several bilaterian and cnidarian iGluRs, some of this being part of Objective 2, and (2) has started an additional line of experimental enquiry that arose from her computational phylogenetics, utilizing various crustacean iGluRs.

Additionally, we had another PhD student start in the lab May 2020, funded by non-ERC means, who has essentially joined the ERC PhD student in exploring Objectives 1 and 2. This additional sub-project has benefited from some of the lessons we had already learnt in Objective 1, and this non-ERC student has used computational phylogenetics and similar electrophysiological experiments to characterize a particular sub-family of bilaterian iGluRs. This work has been productive, has a lot of promise, and the student has begun exploring the molecular changes in this sub-family that led to the strange function of this type of iGluR in vertebrates like us. This work was presented on 20th February 2022 in a poster at the annual meeting of the Biophysical Society (USA).

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 has taken most of the effort of the ERC PhD student, and she has only just begun begun molecular biology experiments aimed at dissecting the molecular basis for the functional/evolutionary hypotheses that we are developing in Objective 1. But she is exploring this along two major lines of investigation: the switch from one major type of iGluR in cnidarians to another type in certain bilaterians; and the evolution of a certain sub-type in crustaceans (described above for Objective 1). We also have a PhD student in the lab through non-ERC means who has begun dissecting the changes during evolution of one particular sub-family of iGluRs in bilaterians, and this looks very promising: he is starting to uncover which parts of these e.g. vertebrate iGluRs have changed to become the unusually non-responsive iGluRs formed by this sub-family of iGluR genes. Another postdoc in the lab funded by other sources has also examined the expression of iGluRs in model organisms with simplified nervous systems, an example is illustrated in the picture attached below.

Objective 3 - Chemical interactions that determine ligand recognition

Ultimately, we hope to inegrate Objective 3 methodolgies - an expanded genetic code with which we can precisely probe the chemistry of neurotransmitter binding to iGluRs - with hypotheses emerging from Objectives 1 and 2. But we started Objective 3 with more familiar rat iGluRs, and the ERC PI and ERC postdoc 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 has now incoroprated several non-canonical amino acids into rat NMDA receptors and performed a lot of regular mutagenesis and pharmacological experiments to better understand the chemistry of rat iGluRs. We are collaborating with a computational biophysics group on this, and they are computationally simulating neurotransmitter binding to NMDA receptors. We hope to wrap this up as a standaline story within a few months, and then we will move on to incorporate this methodlogy into exciting results from Objectives 1 and 2 to start dissecting the chemistry behind the evolution of iGluRs.
Objective 1 has been challenging but has (1) used computational phylogenetics to suggest new hypotheses on iGluR evolution that will be pursued in Objective 2 and (2) has led to the first experimental characterization of iGluRs from certain animals.

Objective 2 is underway, and we are confident that this will reveal how iGluRs evolved in simpler animals to become the mediators of rapid signals in e.g. the human brain.

In Objective 3, we have expanded the genetic code to incorporate non-canonical amino acids into rat NMDA-type iGluRs and our experiments probing the chemistry of neurotransmitter receptor function has revealed that certain long-standinng notions of neurotransmitter binding are perhaps misleading. We hope to finalize this year our story on the chemistry by which rat NMDA-type iGluRs respond to neurotransmitter binding, and then incorporate this methodology into Objectives 1 and 2 to reveal the chemistry that was empoyed by nature during the evolution of different types of iGluRs.
Expression of iGluRs (blue) in an invertebrate with a simplified nervous system.