Periodic Reporting for period 4 - gluactive (Activation Mechanism of a Glutamate Receptor)
Reporting period: 2020-01-01 to 2020-12-31
The project GluActive aims to elucidate the mechanisms of activation of AMPA type glutamate receptors, using functional, structural and computational approaches. This receptor is one of the most abundant in the brain of vertebrates. It is involved in all complex thought. Despite decades of work, bona-fide mechanisms that describe the activation states of this class of receptor are still lacking. It is important for society to understand the activation of glutamate receptors because this information will facilitate investigation of synapses, the acknowledged substrate of learning and memory in the brain. Further, because of their ubiquity, there may be some sense in which understanding the activation of glutamate receptors is useful in brain injury and disease.
Many high-resolution images of the AMPA-type glutamate receptor have recently become available. In these pictures we can see how glutamate attaches to the receptor, and by comparing pictures with and without glutamate, simple models of how glutamate activates AMPA receptors were formed. In this project we seek to extend and organize these pictures and models and test the resulting ideas about how the receptor is activated. We also seek to examine other processes involving the AMPA receptor that are important for passing messages between cells in the brain.
Using advanced computer hardware, we could create a series of movies of glutamate binding to a key brain receptor for the first time. These simulations were accurate to atomic detail and covered nearly 50 µs of real time. Although this time span is almost unimaginably brief, it is highly relevant for the AMPA receptor that is amongst the fastest signaling molecules in the brain of mammals. This set of movies showed that the binding is not random but rather directed. This surprising finding could be confirmed by functional experiments. The intriguing possibility is that these pathways evolved to make the receptor fast. A video is available at: https://twitter.com/AndrewPlested/status/941404076591452160
For some years we have modified receptors so that we can trap them in particular functional states. We now also used a toxin derived from a sea snail to perform a similar trick. When combined with high resolution maps of the receptor, we can understand the geometry that is behind receptor activation. The receptor is composed of four subunits, and each has a binding site for glutamate. Our work has revealed an important relationship between the activity of the receptor and how many different arrangements of the subunits can be attained. There are molecules that activate the receptor less strongly than glutamate, and these tend to push the receptor into “inactive” states. The less strong the activator is, the more different inactive states can be visited. In contrast, glutamate is a good activator because it selects arrangements that have high activity and prevents the receptor from getting lost in unproductive conformations. We started out by looking at receptor activity in ensemles- this is quite easy. But our main aim was to look in detail and single receptors. To do so, we used new analysis techniques and made several studies of activation at the single channel level.
We also used highly calibrated “molecular rulers” to precisely define the geometry of receptors during activation. We found when the receptor is closest to its native form, decorated with helper proteins, it is the most compact – further supporting our idea that high activity corresponds to a narrow set of conformations. This idea has interesting implications for receptors in the brain and we examined the consequences in brain cells as the project progresses , using fluorescent reporters. We also discovered an entirely new form of AMPA receptor in the hippocampus.
Using advanced computer hardware, we could create a series of movies of glutamate binding to a key brain receptor for the first time. These simulations were accurate to atomic detail and covered nearly 50 µs of real time. Although this time span is almost unimaginably brief, it is highly relevant for the AMPA receptor that is amongst the fastest signaling molecules in the brain of mammals. This set of movies showed that the binding is not random but rather directed. This surprising finding could be confirmed by functional experiments. The intriguing possibility is that these pathways evolved to make the receptor fast. A video is available at: https://twitter.com/AndrewPlested/status/941404076591452160
For some years we have modified receptors so that we can trap them in particular functional states. We now also used a toxin derived from a sea snail to perform a similar trick. When combined with high resolution maps of the receptor, we can understand the geometry that is behind receptor activation. The receptor is composed of four subunits, and each has a binding site for glutamate. Our work has revealed an important relationship between the activity of the receptor and how many different arrangements of the subunits can be attained. There are molecules that activate the receptor less strongly than glutamate, and these tend to push the receptor into “inactive” states. The less strong the activator is, the more different inactive states can be visited. In contrast, glutamate is a good activator because it selects arrangements that have high activity and prevents the receptor from getting lost in unproductive conformations. We started out by looking at receptor activity in ensemles- this is quite easy. But our main aim was to look in detail and single receptors. To do so, we used new analysis techniques and made several studies of activation at the single channel level.
We also used highly calibrated “molecular rulers” to precisely define the geometry of receptors during activation. We found when the receptor is closest to its native form, decorated with helper proteins, it is the most compact – further supporting our idea that high activity corresponds to a narrow set of conformations. This idea has interesting implications for receptors in the brain and we examined the consequences in brain cells as the project progresses , using fluorescent reporters. We also discovered an entirely new form of AMPA receptor in the hippocampus.
With a novel combination of methods, including single molecule studies, we investigated the AMPA-type glutamate receptor in unprecedented spatial and temporal detail. So far, we could already understand a lot more about the receptor dynamics and conformations than was previously known. By the end of the project, we hope to relate this to receptors in neuronal cells more directly, using novel labelling and tagging methods based on our findings. There is little wider socio-economic or societal impact, unfortunately. Just an improved understanding of brain function at the molecular level.