Periodic Reporting for period 4 - iGLURs - A NEW VIEW (Exposing nature’s view of ligand recognition in ionotropic glutamate receptors)
Berichtszeitraum: 2023-09-01 bis 2024-08-31
Ionotropic glutamate receptors (iGluRs) are proteins in our brain mediating rapid communication between cells in the brain – “excitatory signalling”. This enables e.g. thoughts, behaviour, and learning. iGluR malfunction is implicated in numerous neurological disorders. Despite access to human and other animal genomes and previous work on iGluR function, we are still unable to look at DNA sequences and accurately predict iGluR function (or malfunction). This project aimed to reach this level of knowledge.
To do so, we aimed to (1) uncover how diverse the iGluR family is; (2) establish how iGluRs evolved to be what they are now; (3) dissect the chemical details of glutamate binding to iGluRs. In other words, "Nature's view of ligand recognition in iGluRs". Techniques included computational phylogenetics, electrophysiological experiments, and chemical biology manipulations of proteins. 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.
Now that the project has finished, we uncovered remarkably diverse function among iGluRs of various animals. For example, we found that some iGluRs are constantly active, with other cells sending signals that “turn iGluRs off”. We established that certain mysteriously inactive iGluRs in the mammalian nervous system evolved out of receptors for GABA, a neurotransmitter that typically activates a different protein in the brain. Furthermore, in some cases, we revealed chemical interactions underlying such function.
To do so, we aimed to (1) uncover how diverse the iGluR family is; (2) establish how iGluRs evolved to be what they are now; (3) dissect the chemical details of glutamate binding to iGluRs. In other words, "Nature's view of ligand recognition in iGluRs". Techniques included computational phylogenetics, electrophysiological experiments, and chemical biology manipulations of proteins. 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.
Now that the project has finished, we uncovered remarkably diverse function among iGluRs of various animals. For example, we found that some iGluRs are constantly active, with other cells sending signals that “turn iGluRs off”. We established that certain mysteriously inactive iGluRs in the mammalian nervous system evolved out of receptors for GABA, a neurotransmitter that typically activates a different protein in the brain. Furthermore, in some cases, we revealed chemical interactions underlying such function.
Objective 1 - Establishing a broad and representative sample of iGluRs
Our computational phylogenetics yielded phylogenetic trees of the iGluR superfamily that (1) recapitulate major relationships suggested by others but (2) shows information from types of animals not previously studied. This revealed interesting patterns in iGluR evolution that we have pursued experimentally. As certain relationships (which iGluR genes belong in which subfamily, and which subfamilies are closely related to which other subfamilies) change from tree to tree, depending on tree algorithm but mostly on which types of animals are included in the dataset, we used different sets of animals depending on which iGluR subfamily we were studying in detail.
Numerous iGluR genes were identified and synthesized, including from comb jellies, sponges, placozoans, sea anemones, various worms, crabs, and starfish. Many genes were challenging to work with in our electrophysiological experiments, especially from distantly (relative to rat/human) related comb jelly and sea anemone genes. However, the ERC PhD student working on this identified ways of boosting iGluR function in our experiments and accumulated much data on the function of these different receptors. We learnt that pursuing such a broad set of genes/proteins, from across iGluRs of all animals, is extremely challenging and focussing on smaller subfamilies in various subprojects is more productive. Some of the interesting results from testing such a broad range of iGluRs was presented in a poster session at the annual meeting of the Biophysical Society (USA), 2023. Several more focussed findings, including novel AKDF-type iGluRs from placozoans, novel delta iGluRs from invertebrates, and new observations on more widely known AMPA-type iGluRs and NMDA-type iGluRs were pursued further in Objective 2.
Objective 2 - Identifying the molecular changes that led to ligand recognition
This can be described in six main lines of work. Firstly, the ERC postdoc’s work on NMDA receptors overlapped surprisingly with the PhD student’s work on epsilon-type iGluRs, and we discovered that one particular amino acid residue in the iGluR family controls receptor activation. This was published in the journal Structure, 2024, as “Constitutive activity of ionotropic glutamate receptors via hydrophobic substitutions in the ligand-binding domain” (see attached “continuous reporting image” for graphical abstract). Secondly, we, including a non-ERC-funded PhD student I supervised on the project, found that delta-type iGluRs in most animals are receptors for the transmitter GABA, in stark contrast to vertebrates where delta-type iGluRs are (a) relatively inactive and (b) receptors for glycine and D-serine, and we identified which amino acid mutations during evolution led to this scenario. This was published in the journal PNAS, 2024, as “Loss of activation by GABA in vertebrate delta ionotropic glutamate receptors”.
The ERC PhD student, ERC postdoc, with help of others in the lab and with other funding now that grant has finished, are finalizing the other four lines of work. The postdoc is finalizing work on the evolution of NMDA-type iGluRs, based on surprising findings on NMDA iGluRs in crustaceans. This was presented in a talk at the annual meeting of the Biophysical Society (USA), 2024. The PhD student (now graduated) is finalizing (a) the evolution of ligand recognition from glycine to glutamate, glutamate to glycine, etc in both AKDF- and epsilon-type iGluRs and (b) the oligomerization of AMPA-type iGluRs in various animals. And another postdoc in the lab has examined the expression of iGluRs in model organisms with simplified nervous systems and finalizing that together with a master’s student in the laboratory. I have presented some of this work in several invited talks, e.g. the EPIC seminar series from the Society for General Physiologists (USA).
Objective 3 - Chemical interactions that determine ligand recognition
In two of the above lines of work we were able to finely dissect certain chemical interactions determining iGluR function. In our 2024 publication as “Constitutive activity of ionotropic glutamate receptors via hydrophobic substitutions in the ligand-binding domain” we incorporated non-canonical amino acids into vertebrate NMDA-type iGluRs to establish chemical interactions underlying channel activation. And in the line of work we are now finalizing, regarding the evolution of ligand recognition from glycine to glutamate, glutamate to glycine, etc in various iGluRs, we have data where non-canonical amino acids were incorporated into several invertebrate and vertebrate iGluRs.
Our computational phylogenetics yielded phylogenetic trees of the iGluR superfamily that (1) recapitulate major relationships suggested by others but (2) shows information from types of animals not previously studied. This revealed interesting patterns in iGluR evolution that we have pursued experimentally. As certain relationships (which iGluR genes belong in which subfamily, and which subfamilies are closely related to which other subfamilies) change from tree to tree, depending on tree algorithm but mostly on which types of animals are included in the dataset, we used different sets of animals depending on which iGluR subfamily we were studying in detail.
Numerous iGluR genes were identified and synthesized, including from comb jellies, sponges, placozoans, sea anemones, various worms, crabs, and starfish. Many genes were challenging to work with in our electrophysiological experiments, especially from distantly (relative to rat/human) related comb jelly and sea anemone genes. However, the ERC PhD student working on this identified ways of boosting iGluR function in our experiments and accumulated much data on the function of these different receptors. We learnt that pursuing such a broad set of genes/proteins, from across iGluRs of all animals, is extremely challenging and focussing on smaller subfamilies in various subprojects is more productive. Some of the interesting results from testing such a broad range of iGluRs was presented in a poster session at the annual meeting of the Biophysical Society (USA), 2023. Several more focussed findings, including novel AKDF-type iGluRs from placozoans, novel delta iGluRs from invertebrates, and new observations on more widely known AMPA-type iGluRs and NMDA-type iGluRs were pursued further in Objective 2.
Objective 2 - Identifying the molecular changes that led to ligand recognition
This can be described in six main lines of work. Firstly, the ERC postdoc’s work on NMDA receptors overlapped surprisingly with the PhD student’s work on epsilon-type iGluRs, and we discovered that one particular amino acid residue in the iGluR family controls receptor activation. This was published in the journal Structure, 2024, as “Constitutive activity of ionotropic glutamate receptors via hydrophobic substitutions in the ligand-binding domain” (see attached “continuous reporting image” for graphical abstract). Secondly, we, including a non-ERC-funded PhD student I supervised on the project, found that delta-type iGluRs in most animals are receptors for the transmitter GABA, in stark contrast to vertebrates where delta-type iGluRs are (a) relatively inactive and (b) receptors for glycine and D-serine, and we identified which amino acid mutations during evolution led to this scenario. This was published in the journal PNAS, 2024, as “Loss of activation by GABA in vertebrate delta ionotropic glutamate receptors”.
The ERC PhD student, ERC postdoc, with help of others in the lab and with other funding now that grant has finished, are finalizing the other four lines of work. The postdoc is finalizing work on the evolution of NMDA-type iGluRs, based on surprising findings on NMDA iGluRs in crustaceans. This was presented in a talk at the annual meeting of the Biophysical Society (USA), 2024. The PhD student (now graduated) is finalizing (a) the evolution of ligand recognition from glycine to glutamate, glutamate to glycine, etc in both AKDF- and epsilon-type iGluRs and (b) the oligomerization of AMPA-type iGluRs in various animals. And another postdoc in the lab has examined the expression of iGluRs in model organisms with simplified nervous systems and finalizing that together with a master’s student in the laboratory. I have presented some of this work in several invited talks, e.g. the EPIC seminar series from the Society for General Physiologists (USA).
Objective 3 - Chemical interactions that determine ligand recognition
In two of the above lines of work we were able to finely dissect certain chemical interactions determining iGluR function. In our 2024 publication as “Constitutive activity of ionotropic glutamate receptors via hydrophobic substitutions in the ligand-binding domain” we incorporated non-canonical amino acids into vertebrate NMDA-type iGluRs to establish chemical interactions underlying channel activation. And in the line of work we are now finalizing, regarding the evolution of ligand recognition from glycine to glutamate, glutamate to glycine, etc in various iGluRs, we have data where non-canonical amino acids were incorporated into several invertebrate and vertebrate iGluRs.
Objective 1
Novel iGluRs have been described with surprising functions, such as (a) activation by the neurotransmitter GABA, and (b) constitutive channel activity in the absence of neurotransmitters (see attached “continuous reporting image” for graphical abstract from latter publication).
Objective 2
Molecular bases for the evolution of the above functions were identified. The use of an evolutionary approach to uncover such molecular bases is novel for the iGluR field, and I think this will have a big impact moving forward.
In Objective 3
One of the above utilized non-canonical amino acid incorporation into iGluRs (an "expanded genetic code") to establish chemical interactions crucial to iGluR function. And non-canonical amino acid incorporation into various invertebrate and vertebrate iGluRs is ongoing, in a line of work that we are still finalizing in the laboratory, dissecting how receptors for glutamate evolve into receptors for glycine, and vice versa. This provides a profound “chemical-scale” insight into the unique evolutionary approach to studying iGluRs described above.
Novel iGluRs have been described with surprising functions, such as (a) activation by the neurotransmitter GABA, and (b) constitutive channel activity in the absence of neurotransmitters (see attached “continuous reporting image” for graphical abstract from latter publication).
Objective 2
Molecular bases for the evolution of the above functions were identified. The use of an evolutionary approach to uncover such molecular bases is novel for the iGluR field, and I think this will have a big impact moving forward.
In Objective 3
One of the above utilized non-canonical amino acid incorporation into iGluRs (an "expanded genetic code") to establish chemical interactions crucial to iGluR function. And non-canonical amino acid incorporation into various invertebrate and vertebrate iGluRs is ongoing, in a line of work that we are still finalizing in the laboratory, dissecting how receptors for glutamate evolve into receptors for glycine, and vice versa. This provides a profound “chemical-scale” insight into the unique evolutionary approach to studying iGluRs described above.