Desensitization of diliganded mouse muscle nicotinic acetylcholine receptor channels

Abstract

Nicotinic ACh receptor channels (AChRs) exposed to high concentrations of ACh adopt 'desensitized' conformations that have a high affinity for the transmitter and no measurable ion conductance. Single-channel currents elicited by 0.1 or 1 mM ACh were recorded from human embryonic kidney (HEK) cells that had been transiently transfected with mouse alpha, beta, delta, and epsilon subunits. On the time scale of approximately 0.1 ms to approximately 1 h, apparent open intervals are described by a single exponential component, and shut intervals associated with desensitization are described by the sum of four or five exponential components. The kinetic behaviour appeared to be stationary and homogeneous. Desensitization rate constants were estimated by kinetic modelling of currents from cell-attached and outside-out patches (where the number of channels in the patch was measured). A single AChR recovered from the longest-lived desensitized state only after approximately 5 min. The occupancy of an AChR for each of the desensitized states was calculated as a function of time after the continuous application of a pulse of saturating ACh. The longest-lived desensitized state accounted for 90 % of the total only after several seconds. The fractional recovery from desensitization (during a 200 ms wash period) decreased as the duration of the desensitizing pulse increased, suggesting that recovery is slower from the longer-lived desensitized states. The free energy landscape for the AChR desensitization reaction in cell-attached patches exhibited an initial destabilization, followed by a plateau region of gradually increasing stability, followed by a deep well.

Figure 10. Desensitization kinetics of AChRs in outside-out patches exposed to 1 mm ACh

A, clean single-channel currents. Example currents (boxes) are shown at increased resolution at the bottom left of this figure. In outside-out patches, there appears to be a dearth of long-duration apparent openings compared to cell-attached patches (see Fig. 2.) B, interval duration histograms. The idealized currents were fitted by a model (above) having one open and five desensitized states. The closed interval components were (τ in ms (fractional amplitude)): 1.43 (0.28), 7.26 (0.21), 41.7 (0.24), 541.6 (0.19) and 2247 (0.08). The rate constants for four patches under these conditions is shown in Table 4. The only significant difference between cell-attached and outside-out patches is that the entry (recovery) rate constants regarding the first (shortest-lived) desensitized state are ∼twofold faster (slower) in the outside-out configuration. C, the microscopic rate constants provide a good description of the macroscopic decay. The whole-patch current during a 2 s pulse of 1 mm ACh (average of five pulses) and the current response computed directly from the rate constants and model shown in B (dotted line) are superimposed. The model predicts the current should decay as the sum of five exponentials (τ in ms): 0.75 (29 %), 4.2 (26 %), 16.1 (50 %), 90.3 (18 %) and 1192 (1.4 %). These values compared well with those obtained by fitting directly the current decay by the sum of four exponentials: 1.6 (9 %), 15.1 (66 %), 69.6 (22 %) and 1143 (3 %). The two fast components predicted from the single-channel analysis are merged into a single (truncated) component in the macroscopic current. The number of channels in the patch was estimated by dividing the peak amplitude immediately following the agonist step by the single channel current. The initial recovery rate constant (D⁵-D⁴) for the patch was normalized by the number of channels (in this patch, 275) to estimate the rate constant for an individual channel. Overall, this value was 0.00374 s⁻¹, which indicates that a single AChR recovers from the deepest desensitized state in ∼267 s or 4.5 min.

Figure 2. Interval duration distributions in a cell-attached patch exposed to 1 mm ACh

A, single-channel currents after the cleanup procedure. The traces at the bottom of the figure show the boxed currents on an expanded time scale. B, interval duration histograms. The idealized currents were fitted by a model (top graph) having one open and five shut components (continuous line). The shut interval components were (τ in ms (fractional amplitude)): 1.02 (0.44), 9.26 (0.21), 45.6 (0.22), 733 (0.06) and 8739 (0.06). The time constants and fractional amplitudes for 10 patches under these conditions are given in Table 1 and Fig. 6. The slowest time constant is predicted to scale with the number of channels in the patch and therefore does not pertain to a single AChR.

Figure 6. Time constants and amplitudes of shut components from all cell-attached patches (1 mm ACh)

For each of four components, the fractional amplitude (a) is plotted as a function of its time constant (τ). Filled symbols are the five-component patches and open symbols are the four-component patches. The τ and a values were calculated for the regions within the dashed boxes (drawn by eye). The third component in the four-component patches is spread between the third and fourth components of the five-component patches. The slowest shut component of each patch was omitted because its parameters are predicted to scale with the number of channels in the patch, which was different for each patch. See Table 1.

Figure 12. The occupancy of diliganded states following a pulse of saturating ACh

A, the star and the linear models were used to calculate the fractional occupancies as a function of pulse duration (all values are given in s⁻¹). All of the states are diliganded; in addition, a diliganded-closed state connected only to the single open state (not shown) was added to each model (k_C→O = 50 000 s⁻¹, k_O→C = 2000 s⁻¹). B, the time evolution of diliganded states. At time zero, the fractional occupancy was 0.96 the open state and 0.04 in the closed state. The time (and fractional occupancy) of the peak for each desensitized state after the onset of the agonist is given in the text. The final desensitized state, D⁵, becomes dominant after ∼0.3 s (star model) and ∼2 s (linear model). The different recovery time courses that have been observed with different durations of agonist pulse (see Fig. 11) are likely to reflect the different agonist dissociation and/or recovery rate constants for desensitized states D¹-D⁵.

Figure 11. The time course of recovery from a desensitizing pulse depends on the duration of the pulse

A, currents from an outside-out patch. The time scale is logarithmic. A pulse of 1 mm ACh was applied for the indicated durations (bars), followed by a 0.2 s wash in ACh-free solution, followed by a second, test pulse of ACh (arrow). The amplitude of the test pulse indicates the number of AChRs that recovered from desensitization during the wash period. The fraction that does not recover increased as the duration of the initial pulse increased. B, the fraction that does not recover as a function of the initial pulse duration. About half of the AChRs failed to recover during the 0.2 s wash when the initial pulse duration was 0.2 ms. The lines are calculated from the estimated occupancy probabilities in the desensitized state at the start of the wash period (Fig. 12). The data are accounted for if 5 % (D⁵) and 69 % (D⁴) of AChRs recover (star scheme), and if 0 % (D⁵), 25 % (D⁴) and 64 % (D³) of AChRs recover (linear scheme), during the 0.2 s wash.

Figure 1. Cleanup procedure

Raw single-channel currents (cell-attached patch, 100 μM ACh, −80 mV; left) typically include ‘noise’ arising from kinetic heterogeneity of ACh receptors (AChRs; a), endogenous currents from non-AChR channels (b), and vagaries associated with the seal (c). Prior to idealization, noisy sections were selected by eye and replaced with the baseline current (right).

Figure 3. Log likelihood analysis of the number of desensitized components

The idealized currents were fitted by models having three to six desensitized components. In each panel, each symbol represents a different patch. In half of the cell-attached (1 mm ACh) patches, the log likelihood improved by at least 10 units when the number of shut components was increased from four to five. Overall, we estimate that five states are required to describe the closed intervals arising from desensitization.

Figure 4. Heterogeneity of desensitization kinetics

A, time constants and fractional amplitudes of intervals within clusters (defined as sequences of > 50 openings containing shut intervals < 3 s), fitted by a three shut/one open component model. There is no evidence of heterogeneity in the fast, intermediate or slow shut components (S₁-S₃). B, shut and open interval duration histograms for all intra-cluster events combined. The continuous line is a fitted by a four-shut component model, and the dashed line is a fitted by a three-shut component model. This patch contained five shut components in all; the slowest component was eliminated from clusters.

Figure 5. Stability of desensitization kinetics

A, time constants and fractional amplitudes of intervals within segments (defined as a continuous 2 min period), fitted by a four shut/one open component model and plotted as a function of the mean time of the segment. Aside from a steady increase in the open time constant (see Fig. 8), there is no clear evidence of non-stationary behaviour in the time constants (S₁-S₄). B, open and shut interval duration histograms for each of the 13 segments. The vertical dashed line shows the open time constant of the first segment, and serves to highlight the drift towards longer open times. The continuous lines in the shut histograms show the fit by four shut components.

Figure 7. Stability of desensitization rate constants

A, clean single-channel currents (cell-attached patch, 1 mm ACh). B, rate constants and interval duration histograms. The idealized currents were fitted by a model (top) having one open and five desensitized states (continuous heavy line in shut interval duration histogram). A fit with a model having only three desensitized states is shown as a light line. C, in order to test the stability of the rate constants, the record was divided into 13, 2 min segments and the intervals within each segment were fitted by a 3-shut component scheme (to allow for the reduced number of intervals in each segment). The rate constants have been normalized by their mean values, as shown. There was no trend in the rate constant estimates, with the exception of the first desensitization step, which slowed by about 1.5-fold over the course of the recording (∼30 min).

Figure 8. Model-independent aspects of AChR desensitization

We are unable to distinguish between the 401 different kinetics schemes (20 without cycles) that contain 1 O and 5 D states. A, all 20 non-cyclic models. B, results of fitting intervals in one patch to the 20 models shown in A. Model 20 is completely uncoupled (star) and model 1 is completely coupled (linear). In each case, there was at least one equilibrium constant (entry/recovery) < 0.1 and at least one > 10. For unknown reasons, the recovery rates (towards open) were less model dependent than the entry rates (away from open).

Figure 9. Desensitization kinetics of AChRs in a cell-attached patch exposed to 100 mm ACh

A, clean single-channel currents. Example currents (boxed areas) are shown at higher resolution at the bottom. B, kinetic model and interval duration histograms. The idealized currents were fitted by a model (above) having one closed, one open and five desensitized states (continuous line). The inset shows the longer-lived interval distribution on an expanded scale. The closed interval components were (τ in ms (fractional amplitude)): 0.15 (0.91), 1.32 (0.03), 15.6 (0.02), 172.6 (0.016), 2946 (0.0079) and 44361 (0.0142). The briefest component is associated with channel activation. The rate constants for five patches under these conditions are shown in Table 3.

Figure 13. The energy landscape of AChR desensitization

The relative free energy along the desensitization reaction coordinate was calculated from the cell-attached patch (1 mm ACh) rate constants using a five-state linear kinetic model (Table 2). The height of the barriers (dotted lines) is arbitrary. The first step is uphill (∼2 k_BT), followed by plateau consisting of three small downhill steps (∼1.5 k_BT each) and, finally, a steep downhill step (∼5 k_BT). As desensitization proceeded, the barrier heights increased.