A critical residue in the α1M2–M3 linker regulating mammalian GABAA receptor pore gating by diazepam
- University of Texas at Austin, Department of Neuroscience, United States
Figures
Figure 1 with 1 supplement
Visual representation of an α1β2γ2 GABAA receptor from cryo-EM map PDB 6X3X.
(A,B) View from the extracellular space (A) and parallel to the membrane plane (B). Bound GABA and DZ are shown as gold and lavender spheres, respectively. The 9' pore residue from each subunit is …
Figure 1—figure supplement 1
Sequence alignment of M2–M3 linker regions for subunits from several members of the pLGIC superfamily.
Position of M2 and M3 helices are indicated above the sequences. The pore gate L9' residue and mutated M2–M3 linker residues are shown in bold.
Figure 2 with 3 supplements
Spontaneous PTX-sensitive and GABA-evoked currents for α1M2–M3 linker alanine substitutions in the gain of function α1L9'Tβ2γ2L background.
(Left) Summary of normalized GABA concentration–response relationships for GABA-evoked currents with the zero current baseline set to the level of spontaneous activity. Solid line is a fit of the …
Figure 2—figure supplement 1
Correction for baseline drift and current rundown.
(A) Raw two-electrode voltage clamp current recording from α1L9'T/K278Aβ2γ2L receptors expressed in a Xenopus laevis oocyte. Responses to 10 second pulses of increasing concentrations of GABA (0.1, …
Figure 2—figure supplement 2
Summary of GABA EC50 and hill slope from fitting Equation 1 to GABA concentration–response relationships as shown in Figure 2 for individual oocytes.
Gray box plots indicate the median and 25th and 75th percentiles. The vertical dashed line is the mean for L9'T.
Figure 2—figure supplement 3
Responses of wild-type α1β2γ2L receptors to 10 second pulses of either 1 mM PTX, 3 μM DZ, or 3 mM GABA.
Figure 3
Ratio of PTX-sensitive to maximal GABA-evoked current amplitude.
(A) Example currents from α1L9'T/V279Aβ2γ2L receptors elicited by 10 second pulses of either 1 mM PTX or 10 μM GABA. (B) Summary of the ratio of PTX-sensitive to maximal GABA-evoked current …
Figure 4 with 1 supplement
Spontaneous PTX-sensitive and DZ-evoked currents for α1M2–M3 linker alanine substitutions in the gain of function α1L9'Tβ2γ2L background.
(Left) Summary of normalized DZ concentration–response relationships for DZ-evoked currents with the zero current baseline set to the level of spontaneous activity. Solid line is a fit of the pooled …
Figure 4—figure supplement 1
Summary of DZ EC50 and hill slope from fitting Equation 1 to DZ concentration–response relationships as shown in Figure 4 for individual oocytes.
Gray box plots indicate the median and 25th and 75th percentiles. The vertical dashed line is the mean for L9'T.
Figure 5
Ratio of maximal DZ-evoked to PTX-sensitive current amplitude.
(A) Example currents from α1L9'T/K278Aβ2γ2L receptors elicited by 10 second pulses of either 1 mM PTX or 1 μM DZ. (B) Summary of the ratio of maximal DZ-evoked to PTX-sensitive current amplitude for …
Figure 6
Spontaneous PTX-sensitive and GABA- or DZ-evoked currents for α1V279D and α1V279W in the gain of function α1L9'Tβ2γ2L background.
(A) Summary of normalized GABA concentration–response relationships for GABA-evoked currents with the zero current baseline set to the level of spontaneous activity (left). Solid line is a fit of …
Figure 7 with 1 supplement
A critical residue in the α1M2–M3 linker regulating DZ’s energetic contribution to pore gating.
(A) A simple model approximating channel gating between closed (C) and open (O) pore states in both unliganded and DZ-bound conditions. (B) Relationship between gating free energy in unliganded (ΔG0)…
Figure 7—figure supplement 1
Single-channel opening for L9'T/V279A in saturating GABA.
(Left) Single-channel recording for an α1L9'T/V279Aβ2γ2L receptor in 100 μM GABA. Dashed lines indicate fully closed (C) and fully open (O) current levels. Bottom trace shows the portion of the top …
Figure 8 with 1 supplement
The mutation α1V279A enhances DZ potentiation of GABA-evoked current amplitudes in a wild-type (WT) α1β2γ2L background.
(A) Normalized GABA concentration–response relationships for WT (circles, three oocytes) and V279A (squares, four oocytes). Solid lines are fits to Equation 1 for all oocytes combined. Fit …
Figure 8—figure supplement 1
Reduction of GABA-evoked currents by the BZD negative modulator FG-7142 for α1β2γ2L (WT) and α1V279Aβ2γ2L (V279A) receptors.
(A) Pair of current responses to 10 second pulses of ~EC20-25 GABA (WT: 10 μM, V279A: 30 μM) followed by a pair of responses to ~EC20-25 GABA + 1 μM FG-7142. (B) Summary of the ratio of peak …
Figure 9 with 1 supplement
A simple MWC model of channel gating largely accounts for the observed effects of α1L9'T and α1V279A via independent and additive effects on the pore-gating equilibrium.
(A) The model depicts channel gating between closed (C) and open (O) states with independent binding of two GABA (G) and one DZ (D) molecule. L is the ratio of closed to open state probabilities in …
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Figure 9—source code 1
MWC simulation code.
- https://cdn.elifesciences.org/articles/64400/elife-64400-fig9-code1-v2.zip
Figure 9—figure supplement 1
Estimated open probability (Po) from the data in Figure 2 (black) overlaid with simulations for the model in Figure 9A (red).
(A) Model fits assuming mutations have identical affinities for GABA (KG = 190 μM, c constrained to 0.003). L9'T: L = 1.5; L9'T/L276A: L = 0.9; L9'T/P277A: L = 1.3; L9'T/K278A: L = 3.9; L9'T/V279A: …
Tables
Table 1
Summary of peak current ratios and ΔΔGDZ for mutations in the L9'T background.
Data are mean ± standard deviation (# oocytes), and individual data points are shown in Figures 3B, 5B, 6C, and 7C.
| IPTX /IGABA-max | IDZ-max /IPTX | ΔΔGDZ (kcal/mol) | |
|---|---|---|---|
| L9'T | 0.36 ± 0.04 (5) | 1.35 ± 0.05 (5) | −0.31 ± 0.04 (5) |
| L9'T/L276A | 0.22 ± 0.09 (4) | 1.62 ± 0.07 (5) | −0.45 ± 0.04 (5) |
| L9'T/P277A | 0.45 ± 0.03 (3) | 1.41 ± 0.17 (5) | −0.46 ± 0.20 (5) |
| L9'T/K278A | 0.20 ± 0.06 (5) | 1.67 ± 0.15 (4) | −0.44 ± 0.08 (4) |
| L9'T/V279A | 0.17 ± 0.04 (4) | 3.30 ± 0.70 (5) | −1.11 ± 0.30 (5) |
| L9'T/V279D | 0.02 ± 0.005 (3) | 2.31 ± 0.08 (3) | −0.52 ± 0.02 (3) |
| L9'T/V279W | 0.16 ± 0.05 (2) | 1.96 ± 0.23 (3) | −0.53 ± 0.10 (3) |
| L9'T/Y281A | 0.25 ± 0.07 (5) | 1.59 ± 0.11 (4) | −0.42 ± 0.07 (4) |
| L9'T/T283A | 0.11 ± 0.03 (5) | 1.71 ± 0.10 (6) | −0.38 ± 0.04 (6) |
