Trapping the activation mechanism of AMPA-type glutamate receptors
Final Report Abstract
We studied the activation of one of the most important neurotransmitter receptors in the brain, the AMPA receptor. We used a combination of structural, biochemical, computation and functional measurements. We elucidated several new arrangements of the structural layer in which the neurotransmitter glutamate binds, which have enabled us to build a preliminary view of glutamate receptor activation. The key advance was the identification, for the first time, of the structural basis for intermediate activation of a glutamate receptor. Further, crosslinks between subunits show the potential to rank existing structures according to their occupancy and kinetics of adoption - a critical insight that is essential for understanding the fast activation and desensitization of the AMPA receptor at synapses. In as yet unpublished work, we were able to identify candidates for the fully active state and other states. The principal application of these results will be in computational and kinetic models of receptor activation, which could be used to understand receptor interactions with the wider set of synaptic proteins, or with therapeutic agents. We were surprised at the extent to which we could discriminate distinct behaviour in functional channels, from different engineered bridges. The process of measuring the adoption of bridges in full-length receptors in their membrane environment, using our fast-perfusion system, took longer than we anticipated in most cases. Measuring crosslinking kinetics across about 10 different conditions for each mutant, and with different exposure times, was quite involved, because each mutant tended to have distinct crosslinking patterns. However, the advantage here was that several bridges yielded conclusive information that enabled us to directly link geometry to activation states. A further unexpected difficulty we encountered was that we were unable to obtain crystal structures of crosslinked receptors in physiological arrangements. Perhaps one rationalisation is that zinc binding sites that are without functional consequence are likely always formed in addition to those that we add rationally. The publication of the intermediate state structure in Neuron was accompanied by a press release from Johns Hopkins Medical Institute, which was picked up by several websites including “Science Daily”, and “Medical News Today”: http://www.sciencedaily.com/releases/2013/08/130807133436.htm http://www.medicalnewstoday.com/articles/264966.php
Publications
- (2013) A conformational intermediate in glutamate receptor activation. Neuron 79: 492-503
Lau AY, Salazar H, Blachowitz L, Ghisi V, Plested AJR, Roux B
(See online at https://doi.org/10.1016/j.neuron.2013.06.003)
