Abstract
The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.
Highlights
Glutamate receptors are essential proteins for transmitting information in the CNS
Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit
We used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligandbinding domain in the presence of several full and partial agonists
Summary
Glutamate receptors are essential proteins for transmitting information in the CNS. Results: The stability of H-bonds at multiple points within the ligand-binding domain varies with the efficacy of the bound agonist. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization. Partial agonists are useful tools for understanding the mechanisms of activation and desensitization (9 –13) because their binding results in differences in cleft closure stability, which can be compared with functional measurements on the intact membrane-bound receptor. We used NMR measurements of hydrogen-deuterium (HD) exchange to probe the equilibrium between the formation and breakage of hydrogen bonds [15] that stabilize GluA2 LBD lobe closure [16] and to illustrate the differences caused by the binding of full and partial agonists. We found that binding of agonist involves formation of new H-bonds within the binding site and changes in the stability of H-bonding outside of the binding site that affect activation, deactivation, and desensitization of the receptor
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