Control of AMPA receptor activity by the extracellular loops of auxiliary proteins
- Humboldt Universität zu Berlin, Germany
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Germany
Figures
Figure 1 with 4 supplements
Modeling and biochemical analysis of AMPA-TARP complexes.
(A) Topology of TARP γ2 (red) and γ8 (blue). Membrane helices numbered from 1 to 4. The first extracellular segment includes a flexible loop (L1, thick section, longer in γ8). Transmembrane helices …
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Figure 1—source data 1
Spot array quantitation.
- https://doi.org/10.7554/eLife.28680.007
Figure 1—figure supplement 1
Loop interactions between TARPs and GluA2.
(A) The middle panel shows TARPs γ2 (red) and γ8 (blue) positioned between equivalent receptor subunits (A and B and C and D). We modeled L1 in two positions, either between the LBD dimer (colored …
Figure 1—figure supplement 2
The negatively charged patch on β4-TM2 loop of γ2 negatively modulates AMPA receptor gating.
(A) The sequence of the β4-TM2 loop of γ2 with the secondary element is shown here. The residues mutated in the negative patch (NP, black box) are marked (diamonds, D88G, E90S and D92G) and the …
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Figure 1—figure supplement 2—source data 1
Rectification indices for negative patch chimera.
- https://doi.org/10.7554/eLife.28680.008
Figure 1—figure supplement 3
Sequence alignment and conservation of TARP loop 2.
TARP sequences (from rat) encompassing the loop 2 are aligned with secondary structure elements on top. Conserved residues are marked by grey boxes and positively and negatively charged residues are …
Figure 1—figure supplement 4
Sequence alignment of γ2 and γ8 constructs.
The sequences of the extracellular regions Loop1 (L1, purple) and Loop2 (L2, cyan) of γ2 (red) and γ8 (blue) are aligned with the secondary structural elements on top. Conserved residues are in …
Figure 2 with 1 supplement
Desensitization properties of γ2 and γ8 L1 mutants.
(A) Representative traces from L1 γ8 in γ2 (red) and L1 γ2 in γ8 (blue) coexpressed with GluA2 in response to a 500 ms pulse of 10 mM Glutamate (kdes = 13 and 55 s−1; Iss = 50% and 30%, …
Figure 2—figure supplement 1
Relief of polyamine block is not affected by loop mutations in γ2 and γ8.
(A) Normalised conductance-voltage plots show that TARP γ2 (red) is better at relieving the polyamine (PA) block of unedited GluA2 receptors (grey) than γ8 (blue). (B) Relief of PA block by γ2 L1 …
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Figure 2—figure supplement 1—source data 1
Rectification indices for electrophysiological recordings of TARP chimeras.
- https://doi.org/10.7554/eLife.28680.013
Figure 3
Desensitization properties of γ2 and γ8 L2 mutants.
(A) Neutralization of L2 in γ2 (γ2 L2_GS, red) decreased Iss, with little effect on γ8 (γ8 L2_GS, blue) (kdes = 50 and 20 s−1; Iss = 5% and 35%, respectively). Representative traces recorded from …
Figure 4
L1 modulates the extent of TARP-mediated superactivation.
(A) Example traces of γ2 wild-type and L1 mutants in response to 7 s application of 10 mM glutamate. During prolonged application of 10 mM Glutamate γ2 induced superactivation of GluA2 receptors, …
Figure 5
Superactivation of γ2 and γ8 L2 mutants.
(A) Neutralizing L2 from γ2 strongly reduced γ2-mediated superactivation (left panel). On this background, L1 from γ8 induced only minimal superactivation (right panel). The grey traces represent WT …
Figure 6
Eliminating L1 and L2 removes modulation by γ2.
(A) Mutation of both L1 and L2 in γ2 (left) and γ8 (right) did not change association of TARPs with AMPA receptors, as assessed by the G-V curve. GluA2 WT is shown in grey. (B) Bar graph summarizing …
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Figure 6—source data 1
Rectification indices for electrophysiological recordings of TARP deletion chimeras.
- https://doi.org/10.7554/eLife.28680.018
Figure 7 with 2 supplements
The LBD-TMD linkers are the key sites for modulation of AMPA receptor gating by TARPs.
(A) Residues in the S1-M1 linker (Gln508, Ser509, and Lys510 represented as yellow atomic spheres) are in close proximity to the L2 of TARPs (L2 of γ2 is shown in red). (B) Residues in the S2-M4 …
Figure 7—figure supplement 1
GluA2 linker mutants do not affect receptor kinetics or assembly with TARPs.
(A) Representative traces from GluA2 linker mutants in response to 500 ms pulses of 10 mM Glutamate. GluA2 WT is shown in grey. (B) Bar graph summarizing the desensitization kinetics and the level …
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Figure 7—figure Supplement 1—source data 1
Rectification indices for electrophysiological recordings of TARPs with GluA2 mutants.
- https://doi.org/10.7554/eLife.28680.022
Figure 7—figure supplement 2
Thermodynamic coupling analysis for AMPAR linkers and γ2 loop 2.
(A) Illustration of the four conditions used to construct the thermodynamic cycle. Receptor (cyan) and γ2 (red) were cotransfected in wild type and mutant forms. (B) Normalised currents in response …
Figure 8
Proposed mechanism of AMPA modulation by TARPs.
(A) Model of a AMPA-γ2 complex in front view (left) and top view (right). Four molecules of γ2 (red) are shown with L1 and L2 colored in magenta and cyan, respectively. L2 is sandwiched between the …
Tables
Table 1
Kinetic properties of wild type and chimeric TARPs and GluA2 linker mutants.
kdes is rate of desensitization, Iss the steady state current expressed as percentage of the peak current and "Superact." the extent of superactivation expressed as the slow increase in steady state …
| Construct | kdes (s-1) | P | Iss (%) | P | Superact. (%) | P |
|---|---|---|---|---|---|---|
| A2 wt | 120 ± 15 (9) | 5 ± 1 | − | − | ||
| γ2 | 60 ± 5 (24) | 25 ± 2 | 7 ± 2 (10) | |||
| γ8 | 40 ± 5 (9) | 25 ± 5 | 30 ± 6 (4) | |||
| γ2 β4 TM2 § | 40 ± 5 (7) | 0.004 | 50 ± 5 | 1 × 10–5 | 17 ± 4 (5) | 0.009 |
| L1 γ8 in γ2 § | 35 ± 5 (30) | 5 × 10–6 | 50 ± 5 | 7 × 10–6 | 27 ± 6 (10) | 0.003 |
| L1 γ2 in γ8 § | 45 ± 1 (28) | 0.34 | 25 ± 3 | 0.86 | 16 ± 1 (16) | 0.001 |
| γ2 ΔL1 § | 60 ± 5 (11) | 0.90 | 15 ± 2 | 0.008 | 6 ± 2 (6) | 0.52 |
| γ8 ΔL1 § | 60 ± 5 (15) | 0.002 | 15 ± 3 | 0.03 | 16 ± 3 (6) | 0.02 |
| γ2 L2_GS § | 65 ± 5 (15) | 0.49 | 5 ± 1 | 1 × 10–6 | 1.3 ± 0.6 (8) | 0.003 |
| γ8 L2_GS § | 25 ± 5 (6) | 0.002 | 40 ± 4 | 0.07 | 12 ± 2 (4) | 0.01 |
| L1 γ8 in γ2 L2_GS § | 10 ± 0.5 (7) | 6 × 10–10 | 45 ± 3 | 6 × 10–5 | 4 ± 2 (6) | 0.19 |
| L1 γ2 in γ8 L2_GS § | 85 ± 5 (6) | 1 × 10–5 | 5 ± 1 | 0.001 | 1 ± 0.7 (6) | 9 × 10–5 |
| γ2 ΔL1 L2_GS § | 80 ± 20 (5) | 0.03 | 2 ± 1 | 4 × 10–4 | 0 (4) | 0.011 |
| γ8 ΔL1 L2_GS § | 60 ± 10 (5) | 0.02 | 10 ± 5 | 0.02 | 3 ± 1 (4) | 0.02 |
| A2 K509A ∆ | 100 ± 5 (5) | 0.34 | 3 ± 0.5 | 0.71 | − | − |
| A2 508GAG510 ∆ | 145 ± 35 (3) | 0.42 | 1 ± 0.5 | 0.27 | − | − |
| A2 781GSG783 ∆ | 110 ± 15 (3) | 0.76 | 2 ± 1 | 0.46 | − | − |
| A2 GAG/GSG ∆ | 150 ± 20 (5) | 0.20 | 2 ± 1 | 0.44 | − | − |
| A2 K509A + γ2 # | 30 ± 10 (5) | 3 × 10–4 | 45 ± 3 | 2 × 10–4 | 5 ± 5 (4) | 0.59 |
| A2 508GAG510 + γ2 # | 70 ± 5 (4) | 0.39 | 10 ± 5 | 0.07 | 0 (3) | 0.03 |
| A2 781GSG783 + γ2 # | 60 ± 5 (9) | 0.60 | 10 ± 1 | 0.001 | 2 ± 0.5 (8) | 0.005 |
| A2 GAG/GSG + γ2 # | 80 ± 5 (8) | 0.01 | 5 ± 1 | 9 × 10–5 | 0 (4) | 0.01 |
| A2 GAG/GSG + L1 γ8 in γ2 # | 12 ± 0.5 (5) | 4 × 10–8 | 30 ± 5 | 0.21 | 2 ± 2 (4) | 0.065 |
| A2 GAG/GSG + γ8 # | 45 ± 2 (5) | 0.30 | 12 ± 3 | 0.03 | 25 ± 5 (5) | 0.37 |
| A2 GAG/GSG + L1 γ2 in γ8 # | 72 ± 5 (5) | 8 × 10–5 | 4 ± 1 | 0.001 | 1 ± 1 (4) | 0.001 |
| A2 GAG/GSG + γ2 L2_GS # | 90 ± 10 (9) | 3 × 10–4 | 2 ± 1 | 5 × 10–6 | 0 (4) | 0.01 |
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Table 1–source data 1
Kinetics and steady state currents from electrophysiological recordings.
- https://doi.org/10.7554/eLife.28680.010
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.28680.024
