The structural arrangement at intersubunit interfaces in homomeric kainate receptors

Abstract

Kainate receptors are glutamate-gated cation-selective channels involved in excitatory synaptic signaling and are known to be modulated by ions. Prior functional and structural studies suggest that the dimer interface at the agonist-binding domain plays a key role in activation, desensitization, and ion modulation in kainate receptors. Here we have used fluorescence-based methods to investigate the changes and conformational heterogeneity at these interfaces associated with the resting, antagonist-bound, active, desensitized, and ion-modulated states of the receptor. These studies show that in the presence of Na + ions the interfaces exist primarily in the coupled state in the apo, antagonist-bound and activated (open channel) states. Under desensitizing conditions, the largely decoupled dimer interface at the agonist-binding domain as seen in the cryo-EM structure is one of the states observed. However, in addition to this state there are several additional states with lower levels of decoupling. Replacing Na + with Cs + does not alter the FRET efficiencies of the states significantly, but shifts the population to the more decoupled states in both resting and desensitized states, which can be correlated with the lower activation seen in the presence of Cs + .

Subunit-selective iGluR antagonists can potentiate heteromeric receptor responses by blocking desensitization

“…More generally, our experiments show that ligand occupancy is an important determinant of AMPA and kainate receptor desensitization, in addition to ligand efficacy (67,68) and structural rearrangements of the LBD dimer interface (42)(43)(44)69). The prevailing view that one agonist-occupied subunit is sufficient to induce efficient desensitization of the tetrameric receptors (26,47) had already been challenged by work on kainate receptors incorporating the high-affinity subunits GluK4 and GluK5 (48)(49)(50) and here is also disproven for other types of kainate receptors, as well as GluA2 AMPA receptor pseudoheteromers.…”

Structural Arrangement Produced by Concanavalin A Binding to Homomeric GluK2 Receptors

“…FRET is ideally suited for such studies, as it can measure distances like a molecular ruler by detecting the extent of energy transfer between a donor and acceptor fluorophore, which are attached to specific sites on the protein of interest. Using the FRET methodology at the single-molecule level allows us to understand the conformational heterogeneity in the protein associated with each of the functional states [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. We have previously used smFRET to investigate the conformations associated with the apo (unliganded), channel open/active, and desensitized states of the GluK2 homomeric kainate receptors [ 34 ].…”

“…Using the FRET methodology at the single-molecule level allows us to understand the conformational heterogeneity in the protein associated with each of the functional states [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. We have previously used smFRET to investigate the conformations associated with the apo (unliganded), channel open/active, and desensitized states of the GluK2 homomeric kainate receptors [ 34 ]. These studies identified the ABD dimer interface as a site showing large-scale changes between active and desensitized states with no significant changes at the ATD interface across the subunits.…”

“…These studies identified the ABD dimer interface as a site showing large-scale changes between active and desensitized states with no significant changes at the ATD interface across the subunits. The ABD dimer interface showed multiples states with varying degrees of decoupling, with the dimer interface being primarily tightly coupled in the resting and active states and shifting toward decoupled states upon desensitization [ 34 ]. This was consistent with the mechanism that was proposed based on cryo-EM structures of the resting and desensitized states [ 6 , 7 , 9 ].…”

“…Prior structural and functional studies have shown that Na + ions stabilize the dimer interface at the agonist-binding domain [ 10 , 35 , 36 , 37 , 38 , 39 ]. Removal of these ions, or their replacement with Cs + , leads to an increase in population of receptor conformations that are decoupled at the dimer interface, and this, in turn, favors inactivation and stabilization of the desensitized state of the receptor [ 34 , 37 ]. Given that Con-A binding favors the coupled dimer interface at the agonist-binding domain, we investigated the ion modulation function of kainate receptors in the presence of Con-A to determine if the two modulators had overlapping conformational control of function.…”