The structural arrangement and dynamics of the heteromeric GluK2/GluK5 kainate receptor as determined by smFRET

Abstract

Kainate receptors, which are glutamate activated excitatory neurotransmitter receptors, predominantly exist as heteromers of GluK2 and GluK5 subunits in the mammalian central nervous system. There are currently no structures of the full-length heteromeric kainate receptors. Here, we have used single molecule FRET to determine the specific arrangement of the GluK2 and GluK5 subunits within the dimer of dimers configuration in a full-length receptor. Additionally, we have also studied the dynamics and conformational heterogeneity of the amino-terminal and agonist-binding domain interfaces associated with the resting and desensitized states of the full-length heteromeric kainate receptor using FRET-based methods. The smFRET data are compared to similar experiments performed on the homomeric kainate receptor to provide insight into the differences in conformational dynamics that distinguish the two functionally. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Keywords: Kainate receptors; Ligand gated ion channels; smFRET.

Figure 1.

Extracellular amino-terminal domain (ATD) and agonist-binding domain (ABD) represented as dimer of dimers in domain swapping configuration (A and D subunits form the dimer at the ABD and A and B at the ATD). Accessible cysteines modified to form cysless constructs for GluK5 and GluK2 subunits are shown as black spheres. Transmembrane domain (TMD) is represented as cartoon structure inside the membrane. A twin-strep tag is attached to the C-terminus of GluK5 subunit shown in green.

Figure 2.

Possible configurations for amino terminal domains of GluK2 and GluK5 subunits in GluK2/GluK5 heterotetramer forming dimer of dimers with distance calculated for K2-K2 subunits and K5-K5 subunits based on homology model for each possible configuration and smFRET experiment.

Figure 3

(A) Full-length structure of the apo state GluK2/GluK5 heteromer (homology model made from PDB 3KG2) with the GluK2 subunits shown in blue and the GluK5 subunits shown in green. Alpha carbon sites at GluK2*−266 are shown as red spheres (B-C) smFRET data for GluK2*−266 sites; with two representative smFRET efficiency traces for individual molecules shown in panel B and cumulative smFRET efficiency traces with observed data (grey) overlaid on denoised data (red) are shown in panel C. Gaussian fits shown in black, blue and green, represents the smFRET efficiency states.

Figure 4.

FRET construct characterization. Representative whole-cell recording for GluK2*−479/GluK5*−471, GluK2*/GluK5*−515, GluK2*−523/GluK5*, GluK2*−266/GluK5* and GluK2*/GluK5*−272.

Figure 5

(A) Full-length structure of the apo state GluK2/GluK5 heteromer (homology model made from PDB 3KG2) with alpha carbon sites at GluK2*−479 (magenta spheres) and GluK5*−471 (red spheres). (B-C) smFRET data for GluK2*−479 and GluK5*−471 sites at the apo state (left panel) and the desensitized state (right panel). (B) Two representative smFRET efficiency traces for individual molecules. (C) Cumulative smFRET efficiency traces with observed data (grey) overlaid on denoised data (red). Gaussian fits shown in black, blue and green, represents the smFRET efficiency states.

Figure 6.

(A) Full-length structure of the apo state GluK2/GluK5 heteromer (homology model made from PDB 3KG2) with alpha carbon sites at GluK2*−523 (red spheres). (B-C) smFRET data for GluK5*−523 sites at the apo condition (left panel) and the desensitized state (right panel). (B) Two representative smFRET efficiency traces for individual molecules. (C) Cumulative smFRET efficiency traces with observed data (grey) overlaid on denoised data (red). Gaussian fits shown in black, blue and green, represents the smFRET efficiency states.

Figure 7.

(A) Full-length structure of apo state GluK2/GluK5 heteromer (homology model made from PDB 3KG2) with alpha carbon sites at GluK5*−515 (red spheres). (B-C) smFRET data for GluK5*−515 sites at apo condition (left panel) and desensitized state (right panel). (B) Two representative smFRET efficiency traces for individual molecules. (C) Cumulative smFRET efficiency traces with observed data (grey) overlaid on denoised data (red). Gaussian fits shown in black, blue and green, represents the smFRET efficiency states.