Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains
- National Institute of Biomedical Imaging and Bioengineering Institutes of Health, National Institutes of Health, United States
- National Institute of Child Health and Human Development, National Institutes of Health, United States
Figures
Figure 1
Sedimentation velocity analysis of the homo-dimerization of GluK1 (A), GluK2 (B), GluK3 (C), and GluK4 (D).
Panel A: Sedimentation coefficient distributions 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 …
Figure 2
Hetero-dimerization of GluK1 and GluK2 ATD.
Panel A: Sedimentation coefficient distribution 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 …
Figure 3
Sedimentation velocity analysis of the hetero-dimerization of GluK1 and GluK2 with GluK4 and GluK5.
Panel A: Sedimentation coefficient distributions 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: …
Figure 4
Homomeric assembly of AMPA receptor ATDs.
Panels A and B: Sedimentation coefficient distributions 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 …
Figure 5
Heteromeric assembly of AMPA receptor ATDs.
Shown are sedimentation coefficient distributions 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 …
Figure 6
Isotherms for heteromeric assembly of AMPA receptor ATDs.
Weighted-average 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 …
Figure 7
Diagram of specific preferential binding of AMPAR and KaiR ATDs.
The Gibbs free energy for various dimers at 20C under the current experimental conditions was calculated from the -values in Table 1 and Table 2. Panel A: Graphic presentation of the …
Tables
Table 1
-values for kainate receptor amino-terminal domain dimer assembly.
https://doi.org/10.7554/eLife.32056.002| DL-GluK1 | DL-GluK2 | DL-GluK3 | DL-GluK4 | DL-GluK5 | |
|---|---|---|---|---|---|
| UL GluK1 | 1.2 µM [0.8, 1.7] | ||||
| UL GluK2 | 1.8 µM [1.5, 2.2] | 163 nM [113, 235] | 9.4 nM [8, 11] | ||
| DL GluK3 | <50 pM* | ||||
| UL GluK4 | 146 nM [116, 184] | 57 nM [46, 71] | >1 mM$ | ||
| UL GluK5 | 18 nM [15,22] | 12 nM [10, 14] | 350 µM% |
-
Values in brackets indicate 95% confidence limits. Abbreviations: UL, Unlabeled protein; DL, DyLight 488-labeled protein; EGFP, EGFP fusion protein. * determination from FDS-SV dilution series. $ determined from SV ABS data. % previously determined from SV ABS data for unlabeled GluK5 (Kumar et al., 2011).
Table 2
-values for AMPA receptor amino-terminal domain dimer assembly.
https://doi.org/10.7554/eLife.32056.007| EGFP-GluA1 | DL-GluA2 | UL-GluA3 | EGFP-GluA4 | |
|---|---|---|---|---|
| UL GluA1 | 28.4 nM [14, 58] | |||
| UL GluA2 | 2.9 nM [1.6, 5.6] | 21.1 nM* [17, 27] | 32.6 nM [19, 59] | |
| UL GluA3 | 8.3 nM [6, 12] | 1.3 nM [1.0, 1.7] | 5.2 µM# [1.7, 14] | 91 nM [60,137] |
| UL GluA4 | 62 nM [15, 218] | 318 nM [145, 730] | ||
-
Values in brackets indicate 95% confidence limits. Abbreviations: UL, Unlabeled protein; DL, DyLight 488-labeled protein; EGFP, EGFP fusion protein. *Similar estimates were obtained separately in previous studies for GluA2 ATD: 24 nM in (Zhao et al., 2016); 16.5–25.4 nM in (Zhao et al., 2013d); 8.3–12 nM in (Zhao et al., 2012). # Value from (Zhao et al., 2012).
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32056.011
