Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains

Abstract

Ion conductivity and the gating characteristics of tetrameric glutamate receptor ion channels are determined by their subunit composition. Competitive homo- and hetero-dimerization of their amino-terminal domains (ATDs) is a key step controlling assembly. Here we measured systematically the thermodynamic stabilities of homodimers and heterodimers of kainate and AMPA receptors using fluorescence-detected sedimentation velocity analytical ultracentrifugation. Measured affinities span many orders of magnitude, and complexes show large differences in kinetic stabilities. The association of kainate receptor ATD dimers is generally weaker than the association of AMPA receptor ATD dimers, but both show a general pattern of increased heterodimer stability as compared to the homodimers of their constituents, matching well physiologically observed receptor combinations. The free energy maps of AMPA and kainate receptor ATD dimers provide a framework for the interpretation of observed receptor subtype combinations and possible assembly pathways.

Keywords: biophysics; glutamate receptors; neuroscience; none; protein interactions; sedimentation velocity; structural biology.

Figure 1.. Sedimentation velocity analysis of the homo-dimerization of GluK1 (A), GluK2 (B), GluK3 (C), and GluK4 (D).

Panel A: Sedimentation coefficient distributions $c(s)$ derived from FDS-SV data of samples with 0.2 nM or 1 nM DyLight 488-labeled GluK1 mixed with 0.3 nM - 40 µM unlabeled GluK1 at the concentrations indicated in the legend (with the asterisk indicating the molecule carrying the fluorophore). Panel B: $c(s)$ traces for an analogous titration series of 1 nM DyLight 488-labeled GluK2 with 2–499 nM unlabeled GluK2 using fluorescence detection, and a 15 µM sample of unlabeled GluK2 determined by absorbance detection at 280 nm. Panel C: $c(s)$ distributions from fluorescence data from DyLight 488-GluK3 at 50 pM - 1.2 µM. Panel D: Analogous $c(s)$ distributions of 0.3–29 µM unlabeled GluK4 detected by absorbance at 280 nm. Panels E and F: Isotherms of signal-weighted average sedimentation coefficients $s_w$ of GluK1 and GluK2, derived from the $c(s)$ distributions in A and B, respectively, and best-fit isotherms of a monomer-dimer model with refined $K_{\mathrm{D}}$ of 1.2 µM for GluK1 E and 160 nM for GluK2 F, respectively. For better visual comparison, $s_w$ is normalized to assign a value of 1.0 to the range from monomer to dimer $s$-value.

Figure 2.. Hetero-dimerization of GluK1 and GluK2 ATD.

Panel A: Sedimentation coefficient distribution $c(s)$ based on FDS-SV data of 1 nM DyLight 488-labeled GluK1 mixed with 0.3–20 µM unlabeled GluK2. A GluK1 control sample without GluK2 (grey) is virtually superimposed by those for 0.3 nM (black) and 1 nM (cyan) GluK2. Panel B: $s_w$ isotherm (normalized to assign a value of 1.0 to the range from monomer to dimer $s$-value) after integration of $c(s)$ traces (circles) and fit with a model for competitive homo- and hetero-association, fixing the two previously measured homo-dimerization constants of GluK1 and GluK2 while refining the $K_{\mathrm{D}}$ for GluK1/GluK2 heteromers to the best-fit value of 1.8 µM.

Figure 3.. Sedimentation velocity analysis of the hetero-dimerization of GluK1 and GluK2 with GluK4 and GluK5.

Panel A: Sedimentation coefficient distributions $c(s)$ determined from analysis of FDS-SV data for titration of 50–200 pM DyLight 488-labeled GluK1 with 0.2 nM - 1.2 µM unlabeled GluK4. Panel B: $c(s)$ distributions for an analogous titration series of 200 pM or 1 nM DyLight 488-labeled GluK1 with 0.2 nM - 5 µM unlabeled GluK5. Panel C: $c(s)$ distributions from analysis of FDS-SV data for titration of 200 pM DyLight 488-labeled GluK2 with 0.2 nM - 1.2 µM unlabeled GluK4. Panel D: Analogous $c(s)$ distributions from FDS-SV data for the titration of 0.5 nM or 1 nM DyLight 488-labeled GluK5 with 0.2 nM - 1.2 µM unlabeled GluK2. Panel E: Isotherms of $s_w$-values for the titration series of GluK1 with either GluK4 or GluK5, fit with a coupled equilibrium model for GluK1/GluK4 or GluK1/GluK5 heterodimer assembly competitive with homo-dimerization of both binding partners, leads to a best-fit heterodimer $K_{\mathrm{D}}$ of 146 nM (95% CI: 116–184 nM) for GluK1/GluK4 and 18 nM (95% CI: 15–22 nM) for GluK1/GluK5. For comparison, the dashed line shows the isotherm for homomeric GluK1. $s_w$ is normalized to assign 1.0 for the range from monomer to dimer $s$-value. Panel F: Analogous to E, isotherms of $s_w$-values for the titration series of GluK2 with either GluK4 or GluK5, fit with a coupled equilibrium model for GluK2/GluK4 or GluK2/GluK5 heterodimer assembly competitive with homo-dimerization of both binding partners, lead to a heterodimer $K_{\mathrm{D}}$ of 57 nM (95% CI: 46–71 nM) for GluK1/GluK4 and 12 nM (95% CI: 6–17 nM) for GluK1/GluK5; the dashed line shows the isotherm for homomeric GluK2.

Figure 4.. Homomeric assembly of AMPA receptor ATDs.

Panels A and B: Sedimentation coefficient distributions $c(s)$ derived from FDS-SV data of a dilution series of 100 pM - 1.2 µM EGFP-GluA1 (A) and 500 pM - 1.2 µM EGFP-GluA4. Panels C and D: Isotherms of $s_w$ for GluA1 (C) or GluA4 (D) fit with a model for monomer-dimer association with a best-fit $K_{\mathrm{D}}$ for GluA1 of 28 nM (95% CI: 14–58 nM) and for GluA4 of 318 nM (95% CI: 145–730 nM). For comparison, the dotted and dashed lines show isotherms for GluA2 and GluA3, respectively. $s_w$ is normalized to assign a value of 1.0 to the range from monomer to dimer $s$-value.

Figure 5.. Heteromeric assembly of AMPA receptor ATDs.

Shown are sedimentation coefficient distributions $c(s)$ for the titrations of 0.2 or 0.5 nM EGFP-GluA1 with 0.2 nM - 1.2 µM unlabeled GluA2 (Panel A), 0.5 nM EGFP-GluA1 1 with 0.2 nM - 3 µM unlabeled GluA3 (Panel B), 0.5 nM EGFP-GluA1 with 0.2 nM - 3 µM unlabeled GluA4 (Panel C), 0.5 or 1 nM DyLight 488-labeled GluA2 with 0.2 nM - 1.2 µM unlabeled GluA3 (Panel D), 0.5 nM EGFP-GluA4 with 0.2 nM - 3 µM unlabeled GluA3 (Panel E), and 0.5 nM EGFP-GluA4 with 1 nM - 3 µM unlabeled GluA2 (Panel F).

Figure 6.. Isotherms for heteromeric assembly of AMPA receptor ATDs.

Weighted-average $s_w$ as a function of concentration were extracted from the sedimentation coefficient distributions shown in Figure 5, and fit with a competitive homo- and hetero-dimerization model using previously determined homo-dimerization parameters of both components as fixed constraints (solid line). Panel A: The analysis of complexes of GluA2 yielded best-fit heterodimer $K_{\mathrm{D}}$-values of 2.9 nM (95% CI: 1.6–5.6 nM) for GluA2/GluA1; 1.3 nM (95% CI: 1.0–1.7 nM) for GluA2/GluA3; and 32.6 nM (95% CI: 19–59 nM) for GluA2/GluA4. For comparison, the dotted line shows the isotherm for homomeric assembly of the GluA2 ATD. (Panel B:) The analysis of heterodimer assemblies not containing GluA2 yields best-fit heterodimer $K_{\mathrm{D}}$-values of 8.3 nM (95% CI: 6–12 nM) for GluA1/GluA3; 61.5 nM (95% CI: 15–218 nM) for GluA1/GluA4; and 91 nM (95% CI: 60–137 nM) for GluA3/GluA4. For comparison, the dashed line shows the isotherm for self-association of GluA3 ATD and the dotted line that of GluA4 ATD. $s_w$ isotherms are normalized to assign 1.0 for the range from monomer to dimer $s$-value.

Figure 7.. Diagram of specific preferential binding of AMPAR and KaiR ATDs.

The Gibbs free energy for various dimers at 20${}^{\circ }$C under the current experimental conditions was calculated from the $K_{\mathrm{D}}$-values in Table 1 and Table 2. Panel A: Graphic presentation of the relationship between the ATDs for AMPAR and KaiR. The color of the big circle (energy cloud) surrounding each pair indicates the free energy level of the dimerization, using the color temperature scale on the right. The width, line type, and color of the arrows emphasizes the stability difference between pairs with exchanged subunits. Panel B and C: Bar graphs of the Gibbs free energy for AMPA (A) and kainate (B) receptor ATD pairs. The dashed lines are visual guides for comparison, at the level of different homodimer free energies.