1. Unitary Properties of AMPA Receptors with Reduced Desensitization
Wei Zhang, Clarissa Eibl, Autumn M. Weeks, Irene Riva, Yan-jun Li, Andrew J.R. Plested, James R. Howe 
Biophysical Journal 
Volume 113, Issue 10, Pages (November 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
Population currents through GluA4 L484Y mutant receptors. (a) Shown here is the current (average of 20 trials) evoked by 500 ms applications of 10 mM glutamate in an outside-out patch containing 100 of GluA4 L484Y mutant receptors. The large steady-state current is roughly half the initial peak current at the start of the application. The decay of the current was fitted with a biexponential function (solid curve; individual components shown as dashed lines). The time constants of each component and their relative amplitudes are indicated. (b) Here is current evoked by a brief (1 ms) application of 10 mM glutamate to another outside-out patch containing GluA4 L484Y mutant receptors. The deactivation decay of the current followed a biexponential time course. The biexponential fit to the decay is superposed on the current trace (solid line). The time constants (and relative amplitude) of the fast and slow components were 1.2 ms (90%) and 8.5 ms (10%). The current trace is the average of 20 consecutive records. (c and d) Recovery from desensitization shows fast and slow components. (c) Example of the results from a two-pulse protocol to measure recovery from desensitization with 10 mM glutamate. (d) Mean ± SE) data from five patches. For each recovery interval, the amplitude of the peak current evoked by the second pulse was divided by the amplitude of the peak current evoked by the first application with which it was paired (peaks were measured from the steady-state current at the end of the first application). The data were fitted with two Hodgkin-Huxley-type components, reflecting the fact that approximately one-third of the receptors recovered substantially faster than predicted from the time-course of the recovery for intervals >20 ms.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
Cyclothiazide reduces population currents through GluA4 L484Y mutant receptors. (a) Shown here are currents evoked by 500 ms applications of 10 mM glutamate in an outside-out patch containing GluA4 L484Y receptors in the absence (black trace) and presence of 100 μM CTZ (red trace). Although CTZ reduces the fade of the current during the application, it also attenuates the amplitude of the glutamate-evoked current. (b) Given here are examples of unitary currents evoked by 500 ms applications of 10 mM glutamate in an outside-out patch containing a single active GluA4 L484Y mutant receptor in control external solution (top) and in the presence of 100 μM CTZ (bottom). In control solution, each trial shown contains bursts of openings separated by long periods (>4 ms) when the receptor is inactive. The long shut periods presumably reflect sojourns in desensitized states. Such long shut periods are still evident in CTZ (bottom three traces), but the bursts tend to be longer in duration. Note the increase in open-channel noise when CTZ is present and the reduction in the mean unitary current. (c and d) Given here are portions of recordings from another one-receptor patch during the continuous application of 10 mM glutamate in the absence (c) and presence (d) of 100 μM CTZ. In both (c and d), the five 5 s segments show 25 s of continuous data. Although the desensitization gaps are shorter in CTZ (d), they are still present (arrows point to examples). Calibration bars in (c and d) are identical. (e) Given here is a portion of the record in (d) on an expanded timescale. The SKM idealization (red) is superposed on the current trace (black). Amplitude levels were estimated from a maximum log-likelihood fit to the entire 5 min record (50 kHz sampling). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. Figure 3
GluA4 wild-type and mutant channels open to multiple conductance levels in the absence and presence of CTZ. (a–d) Shown here are single-channel currents during the continuous application of 10 mM glutamate and amplitude histograms for the entire data set from the same patches for GluA4-wt (a and b) and GluA L484Y (c and d) receptors in normal control solution (a and c) and in 100 μM CTZ (b and d). Data were low-pass filtered at 4 kHz and sampled at 50 kHz. Amplitude histograms of single-channel open events (inward currents at –100 mV) from each patch were fitted with the mixture of four Gaussian components (solid line; individual components shown as dashed lines). Amplitudes were the mean value of data points within individual open events identified by SKM idealization of the entire data set. For the L484Y mutant receptors, but not wild-type GluA4 receptors, CTZ reduced the mean conductance estimates for each of the four open levels detected. Calibrations in (a) apply to all four panels.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

5. Figure 4
Effect of CTZ on currents through GluA2 L483Y receptors. (a) Given here are plots of peak current responses to 10 mM glutamate for patches containing GluA2-wt (left) and GluA2 L483Y (right) receptors. Values in red were obtained during the application of 100 μM CTZ. In some patches, like the example here, after an initial rundown in current amplitude, CTZ potentiated the GluA2-wt response. In other patches, the GluA2-wt peak current declined after CTZ was introduced. In the case of the GluA2 L483Y mutant, CTZ inhibited the current, which was relieved by switching to control solution. (b) Bar plot shows the amplitude of peak currents before and after CTZ application. Wild-type peak currents were, on average, unchanged by CTZ, but the response of the L483Y mutant was reproducibly inhibited (p = 0.027). The solid circles represent the values from the examples plotted in (a). (c) Examples of responses to 10 mM glutamate in the presence and absence of CTZ. Note the much longer decay of the L483Y mutant, which is truncated in the presence of CTZ (red trace). Insets show normalized decays. (d) Decays of the current after glutamate removal were on average ∼3 times longer for the GluA2 L483Y mutant than in any other condition (p = 0.009). Solid circles represent the traces in (c). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

6. Figure 5
Structural comparison of GluA2_LBD_L483Y and GluA2_LBD_L483Y_CTZ. (a) Shown here is structural alignment of the dimeric GluA2_LBD_L483Y in complex with glutamate (purple) and the corresponding complex with CTZ (cyan, PDB: 3TKD). The CTZ molecules, binding between the D1 interface, are shown in sphere representation. (b) Given here are features of the D1 active dimer interface. A single protomer is shown, face-on to the interface, for each complex. Atoms participating in the D1 interface are colored yellow on the surface representation of Chain A of GluA2_LBD_L483Y (left) and GluA2_LBD_L483Y_CTZ (right). Atoms participating purely in CTZ binding are colored orange. Atoms that participate both in the interprotomer interface and CTZ binding are colored dark green. The inset indicates the orientation of protomer and the bound CTZ molecules.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
Cyclothiazide potentiates population and unitary currents through GluA4-Lc receptors. (a) Given here are currents evoked by a brief (1 ms) application of 10 mM glutamate to an outside-out patch containing GluA4 receptors carrying the Lurcher (Lc) mutation (A623T). The traces are averages of 20 trials in the absence (black) and presence (red) of 100 μM CTZ. The complex, prolonged deactivation decays were fitted with three exponential components (the fits, gray, are superposed on the traces). The time constants (relative amplitudes) of the three components are given on the figure. (b) Given here are currents (averages of 20 trials) evoked by 200 ms applications of 10 mM glutamate to another outside-out patch containing GluA4-Lc mutant receptors. In the absence of CTZ (black trace), the current decays to a large steady-state current along a single exponential time-course with a time constant of 11.1 ms. The desensitization decay is not evident in the presence of 100 μM CTZ (red trace). (c and d) Shown here are examples of unitary currents (4 kHz filtering) and amplitude histograms for open-channel currents through GluA4-Lc mutant receptors without (c) and with (d) the addition of 100 μM CTZ to the external solution. The amplitude histograms were fitted (solid line) with the mixture of four Gaussian components (dashed lines) that gave the indicated conductance values. Large conductance events were more frequent in the presence of CTZ. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

8. Figure 7
The L484Y and Lurcher mutations speed activation. (a) Given here is the simplified AMPA receptor kinetic mechanism that shows glutamate binding, LBD closing/opening, and the parallel nature of activation and desensitization. (b) Shown here are population currents (dotted lines, averages of 15–30 trials) evoked by fast applications of 10 mM glutamate for GluA4-wt, GluA4 L484Y, and GluA4-Lc receptors (the applications were 100–500 ms in duration so the vast majority of the responses are off-scale). The currents have been aligned to the onset of the glutamate application and the rising phases of the currents were fitted with H-H equations (solid lines). The fits were used to calculate 10–90% rise-times for each trace, which for the traces shown were 301 μs (GluA4 L484Y), 309 μs (GluA4-Lc), and 384 μs (GluA4-wt). (c) Mean rise-times (bars indicate mean ± SE) for the three GluA4 receptor types (six patches per group). The value for GluA4 L484Y was significantly different from the value for GluA4-wt receptors (∗, p < 0.05) when the three groups were compared with a one-way ANOVA. A two-tailed Student’s t-test comparison of the results for GluA4-wt and GluA4-Lc receptors gave p = 0.066.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

9. Figure 8
Shut times for wild-type and mutant GluA4 receptors. Shown here are examples of shut-time distributions for GluA4-wt, GluA4 L484Y, and GluA4-Lc receptors activated by glutamate without (a) or with (b) CTZ and fit with 3–5 exponential components using log-binned fitting procedures (dashed lines). (c) Shown here are portions (500 ms in length) of activity in one-receptor patches recorded at −100 mV in 100 μM CTZ for GluA4-wt (left), GluA4 L484Y (middle), and GluA4-Lc (right) receptors. Single-channel openings are downward. The examples were selected to illustrate the type of shuttings (at arrowheads) that contribute to the 5–9 ms component seen in all conditions except GluA4-wt without CTZ, which reflects a form of short-lived desensitization.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

10. Figure 9
Burst durations for GluA-wt, GluA4 L484Y, and GluA4-Lc receptors in the absence and presence of CTZ. (a) Shown here are examples of burst-duration distributions for the three GluA4 receptor types. Bursts were defined as series of openings to any open level that were separated by shut periods <4 ms in duration. Each distribution was fitted with two exponential components with the indicated time constants (in milliseconds) and relative amplitudes. (b) Shown here are corresponding distributions from the same three patches obtained in the presence of 100 μM CTZ. The weighted mean burst durations from the fits are increased ∼18-fold for Glu4-wt receptors in CTZ (51.1 vs. 3.66 ms). The presence of CTZ has little effect on burst durations for the L484Y mutation, but bursts for GluA4 L484Y in control solution are longer by more than a factor of 50 compared with GluA4-wt receptors (295 vs. 3.66 ms). Burst durations for GluA-Lc receptors (17 ms) are longer than for GluA4-wt receptors but shorter than for GluA4 L484Y, and the prolongation in the presence of CTZ (12-fold) is greater than for GluA4 L484Y.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions