1. Coupled Control of Desensitization and Gating by the Ligand Binding Domain of Glutamate Receptors 
Anna L. Carbone, Andrew J.R. Plested 
Neuron 
Volume 74, Issue 5, Pages (June 2012)
DOI: /j.neuron
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
The Ligand Binding Domain Determines Glutamate Receptor Recovery
(A) The domain organization of wild-type AMPA (GluA2, red) and kainate (GluK2, blue) receptor subunits and chimeras. The topology of B6P2 and B2P6 chimeras is the same as wild-type channels, with the S1 and S2 segments combining to form the extracellular ligand binding domain (LBD). An orange bar represents the membrane. ATD: amino terminal domain; M1-M4: transmembrane segments.
(B) Wild-type GluA2 receptors recover rapidly from desensitization (left panel, red curve, Hodgkin-Huxley fit with slope 2, krec = 34 s−1). In contrast, wild-type GluK2 receptors recover 2 orders of magnitude more slowly (right panel, blue curve, monoexponential fit, krec = 0.8 s−1).
(C) The B2P6 chimera recovers as rapidly from desensitization as wild-type GluA2 (red curve, Hodgkin-Huxley fit with slope 2, krec = 27 s−1), whereas the inverse chimera, B6P2, has indistinguishable recovery from wild-type GluK2 (blue fitted curve, monoexponential fit krec = 0.5 s−1).
(D) Hodgkin-Huxley type functions fitted to pooled data. For B2P6, krec = 63 ± 6 s−1 for B2P6 (slope = 2, n = 7) and krec = 0.39 ± 0.01 s−1 for B6P2 (slope = 1, n = 10). Dotted and dashed lines show recovery curves for wild-type GluA2 (slope = 2, n = 10) and wild-type GluK2 (slope = 1, n = 14), respectively. Error bars represent standard deviation of the mean (SDM).
(E) Bar plot summarizing exchange of half-recovery times (t50) on a log scale. See also Figures S1 and S2 for further characterization of chimeric receptors.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2 Kinetic Modeling of Chimera Kinetics
(A) Simplified model of glutamate receptor gating. For the simulation shown in (B)–(E), the lifetime of the deep desensitized state (AD2) was varied by systematically changing d2– (red arrow).
(B) Simulated recovery from desensitization profiles for a range of values of d2–. Traces are colored according to values of d2– indicated as in the upper panel.
(C) Simulated currents in response to 10 mM glutamate. Inset shows that all values of d2– produced responses with the same peak open probability. As d2– becomes fast, a steady-state component develops, as for the B2P6 chimera.
(D) One thousand fold variation in d2– does not shift the peak open-probability-concentration relation. Curves for the entire range of values of d2– were indistinguishable, and glutamate EC50 was about 400 μM.
(E) Summary plot shows glutamate potency (EC50, filled circles) and recovery rate (krec) against the value of d2– in each simulation.
(F) Varying the unbinding rates from the desensitized state (kd–, red) and the resting state (k–, green, to enforce microscopic reversibility) gave rise to at most a 10-fold shift in recovery rate, but shifted glutamate potency by more than 2 orders of magnitude (blue circles). Simulations were done on two backgrounds: either low (0.5 s−1, open circles) or high values of d2– (100 s−1, filled circles).
(G) Varying the exit rate from the first desensitized state (red) could not reproduce fast recovery on a background of slow value of d2– (open circles) or slow recovery on a fast value of d2– (filled circles). The unbinding rate from the desensitized state (kd–, green) was altered to impose microscopic reversibility.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3 Two Residues in the LBD Control AMPAR Recovery
(A) Overlay of six paired-pulse records with increasing intervals between pulses. The GluA2 E713T mutant recovered from desensitization (red fitted curve, Hodgkin-Huxley fit with slope 2, krec = 10 s−1) about 4-fold slower than wild-type GluA2 (blue dashed line, 46 s−1, Hodgkin-Huxley function with slope 2). The upper trace indicates the timing of the pulses of 10 mM glutamate.
(B) The GluA2 Y768R mutation slowed recovery about 3-fold and made the recovery monoexponential (red fitted line, krec = 14 s−1). Wild-type GluA2 recovery (blue dashed line) is as in (A).
(C) Combining the single mutations E713T and Y768R had a supra-additive slowing effect on recovery. Eight paired-pulse records are overlaid, illustrating recovery from desensitization that was about 25-fold slower compared to wild-type GluA2 (blue dashed curve). In this patch, the rate of recovery was 1.1 s−1 (single exponential function, red fitted line).
(D) The early phase of recovery for the double mutant on an expanded timescale (same traces as C). Asterisk indicates the response for the double mutant (4% of receptors active) at the same interval by which wild-type GluA2 receptors have fully recovered.
(E) Bar plot summarizing the effects of mutations on half-recovery time (t50). Bars representing mutants with similar recovery behavior to wild-type receptors are colored green. The K716N ΔP717 mutant did not give glutamate-activated currents. Error bars indicate SDM.
(F) A surface plot of the glutamate-bound GluA2 ligand binding domain (PDB code: 1FTJ). Carbon atoms of mutated residues that had only small effects are shown as green spheres. E713 and Y768 are in orange. See also Figure S3 for an sequence alignment of D2 residues and Figure S4 for evidence of thermodynamic coupling between E713 and Y768.
Neuron  , DOI: ( /j.neuron )
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5. Figure 4 Deactivation and Recovery Rates Are Correlated
(A) Glutamate potency for the A2 E713T Y768R double mutant (ET/YR) was intermediate to wild-type GluA2 and GluK2. The EC50 determined from peak currents following a concentration jump (filled circles), was 230 ± 20 μM (n = 5). A dashed line indicates the equivalent curve for wild-type GluK2 receptors, and a dotted line that for wild-type GluA2. Error bars indicate SDM.
(B) Pre-equilibration with low concentrations of glutamate blocks activation with IC50 = 9 ± 1 μM in wild-type GluA2 (open squares, dotted line, n = 3–11 patches per point). The IC50 for GluK2 is much less, 700 ± 80 nM (open circles, dashed line, n = 3–8 patches per point). Glutamate is about 35-fold more potent at blocking activation in the A2 E713T Y768R mutant (IC50 = 240 ± 30 nM, filled circles, solid line, n = 5–7 patches per point) than in wild-type A2.
(C) Mutants of GluA2 that slow recovery also slow the decay of the response to a 1 ms pulse of 10 mM glutamate. Normalized current responses are overlaid for GluA2 wild-type (dashed line), Y768R and E713T Y768R mutants. The upper trace shows the open tip response. Monoexponential fits to the decays are shown, kdeact = 456 s−1 for Y769R (open circles) and 204 s−1 for E713T Y768R (filled circles). The wild-type decay was 1,200 s−1.
(D) A strong correlation (Pearson r = 0.82, 95% confidence interval 0.56 to 0.94) between deactivation and recovery rates for the panel of GluA2 mutants (circles) holds over more than 2 orders of magnitude of recovery rate. Orthogonal distance regression fitted a line with slope 26 ± 3. There was no correlation between the entry and exit rates for desensitization (diamonds, slope 0.01 ± 0.1, r = 0.01, 95% confidence interval −0.5 to −0.5). Wild-type values are indicated with filled symbols.
Neuron  , DOI: ( /j.neuron )
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6. Figure 5 Channel Openings Have Longer Duration in the A2 TR Mutant
(A) Six consecutive records are shown for GluA2 wild-type (left panel) and the A2 TR mutant (right panel) at –80mV. The white bars indicate glutamate application. Black bars indicate sections plotted in panel (B). The asterisk indicates a trace where the piezo stack was actuated but the pipette did not enter glutamate solution. We discarded patches that showed activation from endogenous channels in this condition.
(B) Magnified sections from the traces in (A). In the record corresponding to the TR mutant, some examples of longer open periods can be discerned.
(C) The distributions of open periods (left panel) and shut times (right panel are overlaid for wild-type (black) and A2 TR (gray). See also Figure S5 for 2D histograms of open time against amplitude.
(D) Dot plot showing the mean open period for each patch. Lines indicate the average.
(E) Dot plot of mean conductance weighted by occupancy for each patch.
Neuron  , DOI: ( /j.neuron )
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7. Figure 6
Residues in D2 Also Combine to Control Kainate Receptor Gating and Desensitization
(A) Bar plot summarizing the half recovery times for D2 mutants in GluK2 on a log scale. All mutants were best fit with single exponential recovery functions. The five positions mutated in double, triple, and quadruple mutants are E650, S679, M706, T715, and R769, respectively. Error bars indicate SDM.
(B) Spatial distribution of the ARLE residues (green spheres) in the GluK2 glutamate bound LBD- PDB code 3G3F. A dashed segment indicates a disordered stretch at the C terminus of S2. The site of the GT linker is shown as two black spheres. The bound glutamate is shown as spheres.
(C) Recovery traces from the best recording of the ARLE quadruple mutant are overlaid. Recovery is ∼20-fold faster than wild-type GluK2 (red curve). The upper traces show the piezo command voltages.
(D) Recovery for GluK2 ARLE was best fit with a single exponential function (8.6 ± 0.6 s−1, n = 6 patches). Dotted line shows the recovery of wild-type GluK2 and dashed line that of GluA2.
(E) Mutants of GluK2 that speed recovery also speed the decay of the response to a 1 ms pulse of 10 mM glutamate. Normalized current responses are overlaid for GluK2 (dashed line), LEY (orange fitted curve; kdeact = 810 s−1) and ARLE (green fitted curve; kdeact = 1,440 s−1).
(F) Deactivation and recovery rates for the panel of GluK2 mutants are correlated (diamonds; Pearson r = 0.64; 95% confidence interval 0.23 to 0.86). Orthogonal distance regression fitted a line with slope 490 ± 20. There was little correlation between the entry and exit rates for desensitization (diamonds, slope 16 ± 3, r = –0.01, 95% confidence interval −0.48 to 0.47). Wild-type GluK2 points are indicated with filled symbols.
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
A Simple Mechanism Can Account for the Observed Kinetic Correlations
(A) The modified activation mechanism permits the receptor to transition to the deep desensitized state directly from the open state. Values of the rate constants (per second) were as follows: β = 4,000, d2+ = 150, d2∗+ = 150, d2∗− = 10, k+ = kd+ = 1·107 M−1, k– = 20,000, kd– = 1500, d1+ = 600, d1– = 100, d0+ = 4, d0– = 9. In the simulations shown, the channel closing rate (α, green arrow; range 5.3 to 3,349 s−1) was covaried with d2– (red arrow; range 0.05 to 79 s−1) to ensure microscopic reversibility was obeyed on the loop.
(B) A uniform shift in recovery rate of more than two orders of magnitude results from changes in d2–. In each panel, traces are colored according to values of d2– in the upper panel.
(C) The relaxation at zero glutamate following population of the open state is faster at higher values of d2– (and therefore higher values of α).
(D) The response to a long pulse of 10 mM glutamate is similar for all values of d2–. Low values of d2– and α tended to reduce the peak and steady-state current.
(E) The peak-current concentration relation (normalized to the maximum open probability) is distorted at low concentrations, but glutamate potency shifted only about 7-fold over the entire range of variation in d2– (EC50 range: 130–880 μM). Inset shows the same relation without normalization.
(F) Summary plot of the change in deactivation rate, recovery rate and EC50 with d2–. Coloring corresponds to that in (B).
(G) Values of krec and kdeact derived from simulated currents were strongly positively correlated (open circles) and approximately fit by a power law relation (dotted line, exponent 1.5). The desensitization rate (kdes, filled circles) did not change with krec. Empirically determined values from WT A2 (open diamond), A2 TR (triangle) and A2 DTR (square) are indicated.
Neuron  , DOI: ( /j.neuron )
Copyright © 2012 Elsevier Inc. Terms and Conditions