Desensitization of mouse nicotinic acetylcholine receptor channels. A two-gate mechanism

Abstract

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition (epsilon or gamma), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed.

Figure 9

The time course of recovery from desensitization. The points are from Dilger and Liu (1992) and were obtained using a two-pulse protocol. The first pulse of 100 μM ACh desensitized virtually all AChR in the patch. After a delay, the second pulse was applied to test the fraction of AChR that had recovered from desensitization. The abscissa is the interpulse interval (note the logarithmic scale) and the ordinate is the fractional recovery. The solid line is for the optimal values of Model IV (allosteric model). The initial step of recovery, the dissociation of agonist from desensitized AChR, occurs with a rate constant of ∼23 s⁻¹ (per transmitter binding site), and the final, agonist-independent step of recovery occurs with a rate constant of ∼4 s⁻¹.

Figure 1

Distribution of cluster durations from one patch. A cluster occurs when a single AChR spontaneously recovers from desensitization, and a cluster ends when that receptor again becomes desensitized. Three example clusters are shown (inward current is down). The distribution of cluster durations is described by a single exponential with a time constant of 513 ms. In this patch, there were 63 clusters longer than 100 ms in duration and the mean probability of being open in a cluster was 0.47. The desensitization rate constant, (τ_c P _o)⁻¹, was 3.9 s⁻¹ (−100 mV; 20 μM ACh; 115 mM NaCl).

Figure 2

Cluster properties vs. the concentration of ACh. (top) Example clusters at three different ACh concentrations. (bottom) Between 1 and 100 μM ACh, the probability of being open within a cluster increases and the corrected mean cluster duration (τ_c) decreases. However, the product of these parameters remains nearly constant at ∼300 ms. The mean desensitization rate constant (τ_c P _o)⁻¹ is 3.5 s⁻¹. Each point is the mean of more than two patches, with 5–63 clusters per patch (mean = 27).

Figure 3

The effective desensitization rate as a function of the number of bound ACh molecules. Each symbol is a patch. For each patch, an occupancy probability in unliganded, monoliganded, and diliganded states was calculated from the ACh concentration, the equilibrium dissociation constant (100 μM in 115 mM NaCl and 160 μM in 142 mM KCl), and the gating equilibrium constant according to Model I (see methods). The desensitization rate constant for un- and diliganded AChR was estimated by linear extrapolation to unity occupancy. Diliganded AChR (open plus closed) desensitize at 3.7 s⁻¹ while unliganded AChR desensitize much more slowly, at <0.1 s⁻¹.

Figure 4

The desensitization rate constant, (τ_c P _o)⁻¹, for diliganded AChR does not change with the gating equilibrium constant (θ = β/α; see Model I). θ was varied experimentally by using different receptors, agonists, and membrane potentials. The line is the fit by Eq. 5 with k  ^A₂O _+D = 3.13 s⁻¹ and k  ^A₂C _+D = −0.08 s⁻¹. Over a ∼1,000-fold range in θ, the slope of the line is indistinguishable from zero, indicating that diliganded-open AChR desensitize much faster than diliganded-closed AChR. This suggests that desensitization is a function of the status of the activation gate rather than the occupancy of the binding sites. Each symbol is the average value for a α₂βδε receptor (n patches) (wt [11], αY93F [15], αW149W [4], αG153S [5], εD175N [3], αY198F [10], εE181Q [5], εE184A [4], and I [4]; see Table II) activated under a variety of experimental conditions of agonist (ACh, TMA, CCh), membrane potential (−50, −75, −100, and −130 mV, ) and extracellular salt solution (115 mM NaCl, 140 mM KCl). A total of 61 patches are represented in the plot.

Figure 5

Average cluster properties. Solid lines are Gaussians fitted only to the indicated range of bins. (left) The average desensitization rate constant (τ_c P _o)⁻¹ of the main population is 4.6 s⁻¹. (right) The average cluster frequency of the main population is 0.078 s⁻¹. This value divided by the number of channels in the patch (∼10) is an estimate of the rate constant for recovery from desensitization for diliganded AChR (53 patches; 2–1,000 μM ACh).

Figure 6

Voltage dependence of AChR desensitization. Each point is the diliganded AChR desensitization rate constant, (τ_c P _o)⁻¹, for a single patch (αY93F AChR; four patches). The desensitization rate constant of diliganded AChR does not change significantly between −30 and −130 mV.

Figure 7

Channel block by ACh does not influence AChR desensitization. Each symbol is from one patch (αY93F AChR, −100 mV). (top) Example clusters. (bottom left) The single channel amplitude is reduced at high concentrations because of flickery channel block by the agonist. The equilibrium dissociation constant for ACh block is 1.9 mM. (bottom right) The desensitization rate constant plotted as a function of the fractional occupancy of the pore by the blocker. Channel block by ACh does not influence the desensitization rate constant.

Figure 8

Energetics of the phenomenological model for AChR activation, desensitization, and recovery. (top) The cyclic reaction (C, closed; O, open; and D, desensitized). For simplicity, the two agonist binding steps have been condensed. The numbers are ΔG _O values (k _B T ) and were calculated from the ratios of the rate constants shown in Model IV. The sign of the ΔG _O value pertains to the clockwise direction. (bottom) Graphical representation of the reaction free energies of the states. Unliganded closed is the ground state and [ACh] = 1 M. The A₂D state is the most stable, and only the D state is less stable than the C state. Desensitization has opposite energetic consequences in diliganded (stabilizes) and unliganded (destabilizes) AChR.

Figure 10

A two-gate model for AChR inactivation. The horizontal black bars represent the activation (A) and desensitization (D) gates. The spring represents an interaction between the two gates, which can be ‘relaxed' (compressed) or ‘stressed' (stretched). The square represents the two transmitter binding sites in a low affinity conformation (K  _d = 160 μM in 140 mM KCl), and the circle represents the binding sites in a high affinity conformation (K  _d = 40 nM). The agonist is indicated as being positively charged. The pore is permeable only when both gates are open (upper right). The top row is the activation reaction. At part of the main allosteric transition, the binding sites change affinity, the A gate open/closes, the nature of the AD interaction changes. Below each figure is the state notation, where a bound transmitter molecule is indicated by an A and the status of the gates, closed (C ) or open (O), is given in brackets as [^{A gate} _{D gate}]. The large numbers on the outside of the reaction cycle are ΔG ₀ values in k _B T (sign pertains to the clockwise direction). The smaller numbers on the inside of the reaction cycle break down the ΔG ₀ value into more elemental components. Unliganded AChR can adopt three distinct pore conformations, [^O _C], [^C _C], or [^C _O], that interconvert on the millisecond time scale (spontaneous openings, [^O _O], are extremely rare and have been omitted from the reaction). Unliganded AChR usually reside in the [^C _O] configuration (top left). Proceeding clockwise, activation consists of binding two molecules of ACh (condensed into a single reaction step for clarity) and opening the channel. In the main gating step, A opens and the AD interaction becomes stressed. AChR desensitize when D closes and the interaction relaxes. Upon washout of agonists, AChR recover via the sequence A₂[^O _C] → [^O _C] → [^C _C] → [^C _O]. After ACh dissociates, A closes and the AD interaction is once again stressed. In the final recovery step, D opens and [^C _O] returns to its original relaxed condition.