Opening of glutamate receptor channel to subconductance levels

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate. An ionic current through a single iGluR channel shows up to four discrete conductance levels (O1–O4).

Previously, higher conductance levels have been associated with an increased number of agonist molecules bound to four individual ligand-binding domains (LBDs). Although numerous functional studies support a direct link between ion channel conductance and several independent or nearly independent subunits bound to agonists, this view lacks structural support. Here we determine structures of a synaptic complex of the iGluR subtype AMPA iGluR with the auxiliary subunit γ2 in non-desensitizing conditions (in continuous presence of cyclothiazide, CTZ) with various occupancy rates of the LBDs by glutamate. We show that glutamate binds to LBDs of subunits B and D only after it is already bound to at least the same number of LBDs that belong to subunits A and C.

We used machine-learning approaches to analyse the entire ensemble of the MD-generated conformations of the M3 gate region described by a large set of geometric features, such as pairwise distances between atoms in the neighbouring subunits and across the tetramer, dihedral angles of the residue backbone and side chain, and an area of the pore at T617, A621 and T625 residues. Our structures combined with single-channel recordings, molecular dynamics simulations and machine-learning analysis suggest that channel opening requires agonist binding to at least two LBDs. Conversely, agonist binding to all four LBDs does not guarantee maximal channel conductance and favours subconductance states O1 and O2, with O3 and O4 being rare and not captured structurally. The lack of subunit independence and low efficiency coupling of glutamate binding to channel opening underlie the gating of synaptic complexes to submaximal conductance levels, which provide a potential for upregulation of synaptic activity.

This work was done in collaboration with Dr. Maria Kurnikova group from Carnegie Melon University, Pittsburgh, PA.

Check out Columbia University Irving Medical Center Press release about this work here.