INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Acetylcholine receptors (AChR)¹ are ion channels that open transiently after binding two agonist molecules. In the continuous presence of agonist, AChR become refractory to the stimulus and the cellular response declines. This process, called desensitization, occurs because AChR adopt liganded, stable conformations through which ions cannot permeate (reviewed by Ochoa et al., 1989; Scuka and Mozrzymas, 1992). At the vertebrate neuromuscular junction, desensitization is slow and may not play a significant role in shaping the endplate current or in synaptic depression. However, currents generated by other synaptic receptors often decline rapidly and desensitization is likely to be an important determinant of the amplitude, time-course, and stability of these responses. It is therefore of some physiological importance to understand the molecular events that constitute the desensitization of AChR and other synaptic receptor channels.

Katz and Thesleff (1957) observed that at the frog neuromuscular junction, the steady application of ACh virtually abolished the endplate response within seconds, but upon the removal of the agonist, sensitivity recovered rapidly. Because there was no detectable depolarization during recovery, they proposed a cyclic model for AChR activation and desensitization: agonists bind, AChR open and then "desensitize," and upon washout agonists dissociate and AChR return to their resting condition without reopening. A cyclic reaction scheme implies that unliganded AChR can desensitize.

The affinity of desensitized AChR for agonists (Weber et al., 1975; Boyd and Cohen, 1980; Sine and Taylor, 1982) is ~10,000-fold higher than that of resting AChR (Akk and Auerbach, 1996; Wang et al., 1997), but may be similar to that of open AChR (Colquhoun and Sakmann, 1985). It is important to note that "desensitization" describes a host of inactivation phenomena that may arise from a spectrum of molecular and cellular processes. There are multiple components to the desensitization time course (Heidmann and Changeux, 1979). The main component that was first studied in detail by Katz and Thesleff (1957) occurs on a time scale of seconds, but faster (milliseconds; Sakmann et al., 1980; Magleby and Palotta, 1981; Dilger and Brett, 1990) and slower (minutes; Feltz and Trautman, 1982; Chestnut, 1993) components have been identified. Here, we focus on the component that occurs on the 0.1-1-s time scale.

Several electrophysiological studies have been done regarding the kinetics of this component of AChR desensitization and recovery. Cachelin and Colquhoun (1989; frog muscle) confirmed the cyclic reaction mechanism and speculated that desensitization occurs exclusively from the diliganded, open conformation. They proposed that the rate limiting step to recovery upon washout is the agonist-independent isomerization of the receptor. Dilger and Liu (1992; mouse BC3H1 cells) found that desensitization closely paralleled the open probability of the channel and used the cyclic scheme to estimate molecular rate constants for the desensitization of open AChR (20 s¹) and the recovery of unliganded AChR (3 s¹). Franke et al. (1993; single-channel currents from embryonic mouse muscle) concluded that the recovery from desensitization is rate limited by agonist dissociation rather than an agonist-independent conformational change. They observed that the probability of opening during washout was extremely low, ~10⁴.

Despite a wealth of information on the phenomenology of AChR desensitization, the molecular basis of the reaction remains mysterious. It is not known whether desensitization reflects a global change in the protein structure or more local changes in the conformation of the binding site and/or pore domains. While the above-mentioned kinetic studies have shown that diliganded receptors desensitize faster than vacant receptors, the rate constants for the desensitization of diliganded open vs. closed receptors are not known. This distinction is significant because it illuminates whether desensitization depends on the occupancy of the binding sites or the status of the activation gate. Mutagenesis experiments have not clarified this issue because desensitization is altered by mutations to both binding site residues (Sine et al., 1994) and pore residues (Revah et al., 1991; Weiland et al., 1996; Kuryatov et al., 1997; Milone et al., 1997).

Structural correlates of AChR desensitization have not been clearly identified. Torpedo AChR have been imaged at 9 Å resolution in both the closed (Unwin, 1993) and open (Unwin, 1995) conformations, but only an 18-Å map of desensitized AChR is currently available (Unwin et al., 1988). In this low resolution map, the extracellular domain of the  subunit is seen to be tilted tangentially as a consequence of exposure to carbamylcholine for several minutes. Given that desensitization occurs over minute as well as second time scales, it is likely that the electron diffraction patterns of desensitized Torpedo AChR reflect the slower components of inactivation. Fast inactivation of voltage-gated channels has been attributed to a two-gate ("ball and chain") mechanism (Armstrong et al., 1973; Hoshi et al., 1990), but in AChR it is not known whether the functional distinctions between "closed" and desensitized AChR reflect multiple conformations of a single gate, or different dispositions of multiple gates within the pore.

At the single-channel level, desensitization is manifest as a clustering of channel opening events (Sakmann et al., 1980). Long-lived closed intervals between the clusters reflect times when all AChR in the patch are desensitized. A cluster starts when one AChR recovers from desensitization, and continues with the protein molecule undergoing many cycles of agonist association/dissociation and channel gating. Here, we report desensitization onset and recovery rate constants from the duration and frequencies of single-channel clusters recorded from adult mouse recombinant AChR. The results indicate that the desensitization rate constant is faster when the activation gate is open, and is not a function of the occupancy of the binding sites.

We propose a model in which AChR activation and desensitization reflect the activity of two separate, but interrelated, gates in the ion permeation pathway. In unliganded-closed AChR, the activation gate is usually closed and the desensitization gate is usually open. Binding agonists initiates an allosteric transition (i.e., a global change in structure) in which the binding sites adopt a high-affinity conformation and the activation gate opens. When the activation gate is open, the desensitization gate can close more readily. This configuration (activation gate open and the desensitization gate closed) is very stable. In the two-gate mechanism, the high affinity of a desensitized AChR is simply a consequence of being locked into an activated, but nonconducting, conformation. The recovery process requires agonist dissociation, closing of the main activation gate, and reopening of the desensitization gate. This mechanistic model, which involves only local interactions between the two gates, accounts quantitatively for the phenomenology of AChR desensitization and recovery.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Expression Systems and Electrophysiology

Mouse muscle type nicotinic AChR subunit cDNAs (, , , , or ) were from the laboratories of Drs. John Merlie and Norman Davidson, and were subcloned into a CMV promoter-based expression vector pcDNAIII (Invitrogen Corp., San Diego, CA). The "wild-type"  subunit differed from the sequence in the GenBank database (accession X03986) and had an alanine, rather than a valine, at position 433 (Zhou et al., 1998).

AChR were expressed in human embryonic kidney (HEK) 293 cells using transient transfection based on calcium phosphate precipitation (Ausubel et al., 1992). For muscle type receptors, a total of 3.5 µg DNA per 35-mm culture dish in the ratio 2:1:1:1 (::: or ) was used. The DNA was added to the cells for 12-24 h, after which the medium was changed. Electrophysiological recordings were started 24 h later.

Electrophysiology was performed using the patch clamp technique in the cell-attached configuration (Hamill et al., 1981). The bath was Dulbecco's PBS containing (mM): 137 NaCl, 0.9 CaCl₂, 2.7 KCl, 1.5 KH₂PO₄, 0.5 MgCl₂, 6.6 Na₂HPO₄, pH 7.3. The pipette solution typically contained (mM): 115 NaCl or 142 KCl, 1.8 CaCl₂, 1.7 MgCl₂, 5.4 NaCl, 10 HEPES, pH 7.4. In some experiments, the concentration of KCl was reduced without replacement. In addition, the pipette solution contained the indicated concentration of ACh or other agonist. All experiments were performed at 22-24°C.

Kinetic Analysis

The details of the kinetic analysis methods are described in Akk et al., 1996. Currents were digitized at 94 kHz (VR-10 and VR-111; Instrutech Corp., Great Neck, NY) and were digitally low-pass filtered (Gaussian) using a cutoff frequency (f_c) of 2-7 kHz. Lists of open- and closed-current interval durations were generated via a half amplitude threshold crossing criterion. Clusters were defined as a series of openings separated by closed intervals shorter than some critical duration (_crit). An interval duration histogram of all closures was compiled and fitted by the sum of two to four exponentials. The values of the time constants varied with the concentration of ACh, the expression level of the receptor, the patch area, and in some cases the analysis bandwidth. Typically, there was a fast component (~20-50 µs; sensitive to the bandwidth), an intermediate component that predominated (50-0.05 ms; sensitive to the ACh concentration), and a small, variable, slow component (0.5-5 s; sensitive to the number of AChR in the patch). The initial guess of _crit was more than four times the time constant of the predominant component, or 5 ms, whichever was longer. Clusters were defined accordingly, and an interval duration histogram of all intracluster closures was compiled and fitted by the sum of two to three exponentials. If necessary, the value of _crit was adjusted and the process repeated. The value of _crit depended on the type of receptor, the agonist and its concentration, and the expression levels, but was always at least four times longer than the slowest intracluster closed interval duration component. For example, with wild-type, adult AChR, in 1 µM ACh the time constant of the slowest intracluster closed interval component was ~80 ms and _crit was 400 ms, while in 100 µM ACh, the slowest component was ~0.1 ms and _crit was 5 ms.

The errors associated with cluster definition were not measured for each patch. However, approximate errors can be estimated using typical values for the amplitudes and time constants of the intermediate (a₁ and ₁) and slow (a₂ and ₂) components of the closed interval duration histogram of the entire record. We define r_a = a₁/a ₂, r = ₁/₂, and x = _crit /₁. As shown by Jackson et al. (1983), the fraction of all closed intervals misclassified as being between, rather than within, clusters is a₁e^x and the fraction of all closed intervals misclassified as being within, rather than between, clusters is

These errors will be largest when the agonist concentration is low and the number of AChR in the patch is large. A typical result for a condition with expected large errors (5 µM ACh) was r_a = 10, r = 0.01, and x = 5. Under these conditions, only ~0.7% of the intracluster events and 0.5% of the intercluster events would be misclassified. Moreover, the effect of these misclassifications on the cluster duration and open probability would tend to offset, so the net error in these parameters would be even smaller.

The cluster duration was defined as the time between the first opening and last closing transition. To select for clusters with homogeneous amplitude and kinetic properties, as well as to eliminate isolated openings from the accounting, only clusters >100 ms in duration were included in the calculation of the average cluster duration. If cluster durations are exponentially distributed with an inverse time constant , the relationship between the mean duration _app and  is:

where f (t) is the conditional probability density function, t₁ is the minimum cluster duration, and t ₂ is the maximum cluster duration included in the average. Assuming no upper limit on the cluster duration, the true cluster duration _c (= ¹) is:

(1)

Therefore, to correct for the minimum cluster duration requirement, 100 ms was subtracted from the apparent mean cluster duration for each patch.

The probability of being open within a cluster (P_o) was calculated from intracluster events as the sum of open interval durations divided by the sum of both open and closed interval durations. To insure that an equal number of open and closed intervals contributed to the P_o estimate, the last open interval in the cluster was excluded from this accounting. A mean value of _c and P_o was calculated for each patch.

Analysis of Models

Typically, the activation of an AChR requires the association of an agonist molecule to each of the two transmitter binding sites, followed by a concerted channel gating event. These events are encoded in the standard model for AChR activation (del Castillo and Katz, 1957; Magleby and Stevens, 1972):

View larger version (4K): [in this window] [in a new window]

where A is the agonist concentration, k₊₁ and k₊₂ are the agonist association rate constants, k₁ and k₂ are the agonist dissociation rate constants,  is the channel opening rate constant of a diliganded receptor, and  is the channel closing rate constant of a diliganded receptor.

Steady state occupancy probabilities (P) as a function of the agonist concentration calculated according to Model I are:

(2)

where K₁ and K₂ are the receptor equilibrium dissociation constants, and  is the gating equilibrium constant (/). If the two transmitter binding sites have approximately the same equilibrium dissociation constant (K _d), then Eq. 2 can be simplified with K₁ = 0.5 K _d and K₂ = 2 K _d. Although many studies show that the equilibrium dissociation constants for the two sites are markedly different for some antagonists, analyses of adult mouse AChR indicates that the K _ds for ACh are nearly equivalent at the two sites (Akk and Auerbach, 1996; Wang et al., 1997).

With this simplification, the occupancy probabilities can be related to the probability of being open within a cluster:

(3)

Linear fits were done using Origin (Microcal Software, Northampton, MA). Interval duration histograms and dose-response profiles were fit using NFIT (Island Software, Galveston, TX). The optimization of the rate constants for recovery from desensitization upon washout (see Fig. 9) was carried out by solving the differential equations for the reaction using Scientist (MicroMath, Salt Lake City, UT). Fitted parameters are reported as mean ± SD. Eq. 7 was derived using the symbolic math program Maple (Waterloo Maple, Inc., Waterloo, Ontario, Canada).

View larger version (9K): [in this window] [in a new window]   Fig. 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 s1 (per transmitter binding site), and the final, agonist-independent step of recovery occurs with a rate constant of ~4 s1.

Mutant AChR

The  subunit mutants shown in Table II were a kind gift from Dr. Steven Sine (Mayo Foundation, Rochester, MN). The  subunit mutants were made using overlap PCR as described in Higuchi (1990). The final construct was completely sequenced in the region between the ligation sites.

Drugs

All reagents, including acetylcholine chloride, carbamylcholine chloride, and tetramethylammonium iodide were purchased from Sigma Chemical Co. (St. Louis, MO).

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Desensitization Versus the Agonist Concentration

Fig. 1 shows example clusters elicited by 20 µM ACh. In this patch, there were 63 clusters >100 ms in duration, and the apparent mean cluster duration was 590 ms. After applying a correction for the minimum cluster duration, the mean cluster duration estimate, _c, was 490 ms. The distribution of cluster durations was fitted by a single exponential function with a characteristic time constant of 513 ± 64 ms. There was a reasonably good agreement between the corrected mean cluster duration and the time constant obtained by fitting the distribution.

View larger version (26K): [in this window] [in a new window]   Fig. 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, (c Po)1, was 3.9 s1 (100 mV; 20 µM ACh; 115 mM NaCl).

Most patches had too few clusters to allow fitting of the cluster duration distribution, thus _c was used as the estimate of the cluster duration time constant. There was substantial scatter in the _c estimates, in part because of the small number of clusters measured in each patch. However, some of the variance may arise from nonstatistical reasons, as almost every quantitative electrophysiological study of AChR desensitization has noted considerable variance in the parameters (see Bufler et al., 1993).

Fig. 2 shows the properties of clusters elicited by 1-500 µM ACh. The probability of being open within a cluster increases with the ACh concentration because the time required to bind agonist decreases, leading to shorter closed interval durations. Over the same concentration range, _c decreases ~100-fold. However, the product _cP_o remains relatively constant, with an average value of 285 ms.

View larger version (22K): [in this window] [in a new window]   Fig. 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 (c) decreases. However, the product of these parameters remains nearly constant at ~300 ms. The mean desensitization rate constant (c Po)1 is 3.5 s1. Each point is the mean of more than two patches, with 5-63 clusters per patch (mean = 27).

The constancy of the _cP_o product with respect to the ACh concentration suggests that receptors desensitize primarily from a diliganded state. To quantify the extent to which states of Model I serve as gateways to desensitized states, the inverse of _c at different ACh concentrations was plotted as a function of the probability a receptor occupies unliganded, monoliganded, and diliganded states (Fig. 3). These probabilities were computed according to Model I (Eq. 2) using the salient equilibrium constants: K _d 100 µM (in 115 mM NaCl) and   50 (at 100 mV).

View larger version (16K): [in this window] [in a new window]   Fig. 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 s1 while unliganded AChR desensitize much more slowly, at <0.1 s1.

In general, the cluster duration distribution will have as many components as states within a cluster. However, if transitions between these states are fast compared with desensitization, the distribution will tend towards a single exponential with:

(4)

where P_i is the steady state probability of occupying state i and kⁱ_+D is the desensitization rate constant for that state. Thus, if a state is the predominant outlet to a desensitized state, then the inverse of the cluster duration should increase approximately linearly with occupancy of that state, with a proportionality constant kⁱ_+D. Moreover, for each class of AChR, the extrapolated value of _c¹ at P_i = 1 is an estimate of the intrinsic desensitization rate constant from that class.

Fig. 3 shows that with ACh as the agonist, the effective desensitization rate is positively correlated with the occupancy of diliganded states, confirming the previously established result that desensitization occurs mainly from diliganded states. From these data, very little can be deduced about desensitization from monoliganded states, as these are occupied with only a small probability. The P values for un- and diliganded AChR span a wide range, allowing estimates of k_+D obtained by extrapolation to P = 1 for these classes. For the diliganded points, the fitted straight line has slope of 3.5 ± 0.5 s¹ and an ordinal intercept of 0.24 ± 0.60 s¹, yielding an extrapolated k ^A₂O_+D for diliganded receptors of 3.7 ± 1.1 s¹. For the unliganded points, the fitted straight line slope of 3.6 ± 0.9 s¹ and an ordinal intercept of 3.5 ± 0.3 s¹, yielding an extrapolated value of k^C_+D (unliganded AChR) = 0.1 ± 1.2, which is indistinguishable from zero. The values are scattered and the desensitization rate constants estimated from this analysis are not precise. However, from this analysis we conclude that diliganded AChR desensitize much more rapidly than unliganded receptors.

Desensitization from Open and Closed Diliganded States

Although it is clear that the desensitization of diliganded AChR is relatively fast, the desensitization rate constant of open vs. closed diliganded AChR have not been separately estimated. That is, it is possible that desensitization occurs rapidly from the rarely occupied A₂C state, as was assumed by Naranjo and Brehm (1993), or more slowly from the frequently occupied A₂O state, as was assumed by Cachelin and Colquhoun (1989), Dilger and Liu (1992), and Franke et al. (1993). Making this distinction is important because it illuminates whether it is the number of bound agonists or the status of the activation gate that influences the desensitization rate constant.

To make this separation, the inverse of _cP_o was plotted as a function of the gating equilibrium constant ( = /, from Model I). Because desensitization occurs mainly from diliganded AChR, we combine Eqs. 3 and 4 to produce:

(5)

By examining the relationship between the product _cP_o (measured on a cluster-by-cluster basis) and (estimated from dose-response curves or from single-channel kinetic analysis), the values of k ^A₂O_+D and k ^A₂C_+D can be separately estimated and a determination can be made whether diliganded closed and/or open states are outlets to desensitized states.

For wild-type, adult mouse AChR activated by ACh,   50 (Sine et al., 1995; Wang et al., 1997); i.e., the fractional occupancy of the diliganded closed state is ~0.02 that of the open state. Three experimental manipulations were used to change : different agonists, different membrane potentials, and mutations (see Table II). With these manipulations, cluster durations and open probabilities could be examined over a wide range of  values.

Fig. 4 shows that the inverse of the _cP_o product is approximately independent of the gating equilibrium constant, . The fit of these data by Eq. 5 yields k ^A2O_+D = 3.18 ± 0.33 s¹ and k ^A2C_+D = 0.08 ± 0.21 s¹. The desensitization rate constant for diliganded closed AChR, like that of unliganded closed AChR, is much slower than that of diliganded open AChR and is statistically indistinguishable from zero.

View larger version (7K): [in this window] [in a new window]   Fig. 4.   The desensitization rate constant, (cPo)1, for diliganded AChR does not change with the gating equilibrium constant ( = /; see Model I).  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 s1 and k A2C+D = 0.08 s1. Over a ~1,000-fold range in , 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 2 receptor (n patches) (wt [11], Y93F [15], W149W [4], G153S [5], D175N [3], Y198F [10], E181Q [5], E184A [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.

We conclude that the value of (_cP_o)¹ is a direct measure of the rate constant of desensitization of diliganded open AChR. To obtain a more global estimate of desensitization and recovery (in the steady presence of agonist) rate constants, (_cP_o)¹ and the cluster frequency were measured in 61 patches (115 NaCl or 142 mM KCl in the pipette) activated by 2-1,000 µM ACh (Fig. 5). Although the values are scattered, the main population of patches is centered around values of k ^A2O_+D = 4.6 s¹. There was even greater scatter in the recovery rate for diliganded receptors mainly because this parameter is a linear function of the number of AChR in the patch. Nonetheless, there was a predominant population of cluster frequencies centered around 0.08 s¹. Because we studied cell-attached patches, we could not estimate the number of AChR in each patch. In outside-out patches from embryonic mouse muscle, Franke et al. (1993) found that there were 10-20 AChR per patch. If we assume that in our experiments there is an average of ~10 AChR in a cell-attached patch, then the recovery rate constant (in the continuous presence of agonist) of a single AChR is ~0.01 s¹; i.e., that it takes ~2 min for a diliganded AChR to recover from desensitization.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Average cluster properties. Solid lines are Gaussians fitted only to the indicated range of bins. (left) The average desensitization rate constant (cPo)1 of the main population is 4.6 s1. (right) The average cluster frequency of the main population is 0.078 s1. 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).

To summarize, the results presented thus far indicate that desensitization mainly proceeds from a single outlet state, A₂O, with a rate constant of ~4 s¹, and a diliganded AChR recovery rate constant of ~0.01 s¹.

Effects Of Agonists, Voltage, Ions, and Channel Block

Because desensitization occurs mainly from diliganded, open receptors, k ^A₂O_+D (from now on called simply k^O_+D) can be readily estimated as (_cP_o)¹; i.e., without detailed knowledge of the activation rate constants. The value of k^O_+D was determined for AChR activated by carbamylcholine or tetramethylammonium. AChR activated by these ligands open ~10× slower than those activated by ACh (Zhang et al., 1995). The results (Table I) indicate that the desensitization rate constant does not vary significantly between these agonists.

The properties of the mutant receptor Y93F desensitization were examined at four different voltages. The voltage dependence of the opening and closing rate constants have been determined for this mutant (Auerbach et al., 1996). Fig. 6 shows that for these mutant AChR the product k^O_+D is approximately constant between 55 and 130 mV. The intrinsic rate constant of AChR desensitization is not sensitive to the membrane potential.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Voltage dependence of AChR desensitization. Each point is the diliganded AChR desensitization rate constant, (cPo)1, for a single patch (Y93F AChR; four patches). The desensitization rate constant of diliganded AChR does not change significantly between 30 and 130 mV.

We next investigated whether the ionic composition of the current influences desensitization. In wild-type AChR (V_m = 100 mV), k^O_+D was 5.2 ± 0.6 s¹ in 115 NaCl (n = 21), 3.3 ± 0.6 s¹ in 140 KCl (n = 7), and 5.7 ± 1.6 s¹ in 140 mM CsCl (n = 5). With regard to these monovalent species, the extracellular ionic composition does not have a significant effect on AChR desensitization.

At millimolar concentrations, ACh enters the pore region and transiently (<20 µs) occludes the flow of Na⁺ and K⁺ until it unbinds (Sine and Steinbach 1984; Ogden and Colquhoun, 1985; Maconichie and Steinbach, 1995). The effects of occupancy of the pore by a channel blocker, ACh, are shown in Fig 7. Under our experimental conditions, channel block by ACh is too fast to be resolved as discrete gaps and is instead manifest as a reduction in the mean open channel current amplitude and an increase in the apparent open channel lifetime. The effect of channel block by ACh was examined in an Y93F AChR. The affinity of the pore of this mutant for ACh was estimated from the single-channel current amplitudes (i) at different ACh concentrations (A): i = i ₀/(1 + A/K_block), where i ₀ is the current amplitude in the absence of blockade and K_block is the equilibrium dissociation constant for the ACh-pore interaction. At a membrane potential of 100 mV, K_block = 1.9 mM. _cP_o was measured at this voltage in Y93F AChR activated by 0.5-8.0 mM ACh. In Fig. 7 (bottom right), the desensitization rate constant is plotted as a function of the fractional occupancy of the pore by ACh (f  ): f = (1 + K_block/A)¹.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Channel block by ACh does not influence AChR desensitization. Each symbol is from one patch (Y93F 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.

Although the data are scattered, over the fractional occupancy range of 0.18-0.77 there is no change in the desensitization rate constant (mean = 2.3 s¹). Thus, the occupancy of the pore by ACh apparently does not affect k^O_+D. AChR desensitize from either the blocked or unblocked states with essentially the same rate constant.

Effect of Subunit Composition and Mutations

Embryonic AChR contain a  subunit in place of the  subunit that is present in adult-type receptors (Mishina et al., 1986). In six patches with embryonic-type AChR (5-100 µM ACh, 100 mV), the mean value of k^O_+D was 4.6 ± 1.6 s¹ (range = 3.3-8.0 s¹). This value is not significantly different than the desensitization rate constant of adult-type AChR, indicating that the  vs. subunit does not have a significant effect on k^O_+D.

Several AChR having mutations near the binding site were examined, and the results are shown in Table II. All of the mutations (on the  and  subunits) lowered the gating equilibrium constant, usually by slowing the channel opening rate constant, and many increased the equilibrium dissociation constant for ACh. However, none of the mutations had a measurable effect on the desensitization rate constant. That k^O_+D is neither agonist dependent nor sensitive to mutations that otherwise alter binding and gating suggests that conformational changes at the binding sites are not rate limiting to the desensitization of diliganded AChR.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The most significant experimental finding is that desensitization occurs much faster when the AChR activation gate is open compared with when it is closed. The   molecular rate constant for the desensitization of closed AChR is slow for both unliganded and diliganded species, which suggests that in itself the occupancy of the binding sites is essentially irrelevant to the desensitization process. We conclude that experimental manipulations that alter the macroscopic desensitization rate, such as the agonist concentration, membrane potential, temperature, and certain binding site mutations, do so by changing processes that influence the probability that the AChR activation gate is open, rather than the desensitization rate constant per se.

We emphasize that our experiments and analyses only address the molecular mechanism of the component of desensitization that occurs on the second time scale, and that the physical bases of faster and slower components of desensitization may be quite distinct from those we propose.

Phenomenological Model

Under our experimental conditions, un- and monoliganded open states can be ignored because they are occupied with a low probability. In addition, we have found that desensitization rarely occurs from diliganded, closed receptors. For a useful, if phenomenological, model for AChR operation (modified from Katz and Thesleff, 1957; Cachelin and Colquhoun, 1989), see Model II. A is the agonist and C, O, and D represent closed, open, and desensitized AChR, respectively, k₊ and k are the agonist association/dissociation rate constants for a closed AChR,  and  are the opening/closing rate constants for diliganded AChR, k^O_+D and k^O_D are the desensitization/recovery rate constants for diliganded-open AChR, j₊ and j are the agonist association/dissociation rate constants for a desensitized AChR, and k^C_+D and k^C_D are the desensitization/recovery rate

View larger version (7K): [in this window] [in a new window]

The following equilibrium constants can be defined: K is the equilibrium dissociation constant of the closed conformation (= k/k₊), J is the equilibrium dissociation constant of the desensitized conformation (= j/j₊),  is the channel gating equilibrium constant (= /), D_o is the desensitization equilibrium constant of the doubly liganded, open conformation (= k^O_+D/k^O_D), and D_c is the desensitization equilibrium constant of the vacant, closed conformation (= k^C_+D/k^C_D).

There are two paths by which AChR can recover from desensitization: a diliganded receptor can directly reopen (A₂D  A₂O; equilibrium constant D_O), or the agonist can dissociate and the protein can return to the closed, resting state (D  C; equilibrium constant D_C). From Model II, and assuming detailed balance:

(6)

For recombinant mouse adult-type AChR: K = 160 µM (in 142 mM KCl; Akk and Auerbach, 1996) and J = 0.04 µM (Sine et al., 1995; from binding profiles of proadifen-treated receptors). At 100 mV,   50 (Sine et al., 1995; Akk and Auerbach, 1996). Therefore, from Eq. 6, D_O/D_C = 3.2 × 10⁵.

D_O can be estimated from the kinetic parameters. The rate constant for the A₂O  A₂D transition is 4 s¹. The affinity of desensitized AChR is high and in the continuous presence of agonist the long closed intervals between clusters reflect mainly the A₂D  A₂O recovery pathway. We estimate that the rate constant for this process is ~0.01 s¹. From the ratio of these rate constants, D_O 400. In the presence of a high concentration of agonist, an AChR is desensitized ~99.8% of the time.

With this value for D_O, we use Eq. 6 to calculate that D_C 0.0013, which is only approximately four times larger than the estimate obtained from fitting reaction schemes to binding profiles (Sine et al., 1995; D_C = 3 × 10⁴) and is close to the value obtained from electrophysiological measurements of embryonic mouse muscle AChR (Franke et al., 1993; D_C = 10³). In the absence of ACh, we estimate that only ~1 receptor in ~700 is desensitized.

We can convert these equilibrium constants into free   energy differences using the relationship G₀ = RT ln(K ). When the channel is open and diliganded, desensitization produces a net stabilization of the system of 6.0 k_BT. When the channel is unliganded, the recovery from desensitization produces a net stabilization of the system of 6.7 k_BT. Desensitization has nearly opposite energetic consequences when the activation gate is open compared with when it is closed.

Other workers have used a double-pulse protocol to measure the time course of AChR recovery from desensitization (Katz and Thesleff, 1957; Cachelin and Colquhoun, 1989; Dilger and Liu, 1992; Franke et al., 1993). Upon the removal of acetylcholine, A₂D receptors return to the C state with a time constant of ~300 ms (see Fig. 9). There are no channel opening events during the interpulse interval, indicating that in the absence of agonist, recovery is essentially exclusively via the D  C transition. For the reaction sequence for recovery in the absence of agonist,

View larger version (3K): [in this window] [in a new window]

According to this scheme, the recovery time course in the absence of agonist (A₂D to C) should be the sum of three exponential components. Under the condition that k^C_+D << k^C_D:

(7)

where C(t) is the fraction of AChR in state C at time t. The experimentally determined recovery time course (Dilger and Liu, 1992) was fitted by Eq. 7. The results, shown as the solid line in Fig. 9, were j = 23.0 ± 4.1 s¹ and k^C_D = 4.2 ± 0.4 s¹. Dissociation is only five times faster than the agonist-independent recovery step, and both processes contribute to the recovery time course.

The desensitization and recovery parameters for recombinant AChR are very similar to those for AChR expressed in BC3H1 cells (Dilger and Liu, 1992). This demonstrates that desensitization is determined by factors that are intrinsic to the AChR pentameric complex. Our estimate of the A₂O  A₂D rate constant (4 s¹) is significantly lower than the estimates of Dilger and Liu (1992) and Franke et al. (1993; embryonic mouse muscle) who measured this rate constant to be ~20 s¹. This difference cannot be traced to the  vs.  subunit difference between the preparations because adult and fetal AChR show similar desensitization kinetics. It is possible that the difference may arise from a difference between outside-out and cell-attached patches, or that posttranslational events can influence AChR desensitization and may differ in native and human embryonic kidney expression systems.

We can estimate the ACh association rate constant to desensitized AChR from the equilibrium dissociation constant (40 nM) and the dissociation rate constant (~20 s¹). The association rate constant of ACh to desensitized AChR is fast, ~5 × 10⁸ M¹ s¹. This value is similar to the ACh association rate constant for dansyl-C6-choline to desensitized Torpedo AChR at 0°C (10⁸ M¹ s¹; Heidmann and Changeux, 1979) as well as to nondesensitized (low affinity binding sites) adult mouse AChR (10⁸ M¹ s¹; Akk and Auerbach, 1996). We conclude that desensitization hardly changes the association of ACh to the transmitter binding sites. The increase in affinity that accompanies desensitization is almost completely due to an ~800-fold decrease in the ACh dissociation rate constant. This indicates that when the binding sites are in their high affinity configuration, each ACh molecule is ~6.7 k_BT more stable than when the binding sites are in their low affinity configuration.

The optimal rate constants of the phenomenological model of AChR activation, desensitization, and recovery rate constants (142 KCl, 100 mV, 22°C, ACh) are shown

View larger version (10K): [in this window] [in a new window]

The reaction free energies according to this scheme are summarized in Fig. 8. The A₂D state is 27.3 kT more stable than the resting C state. During recovery, AChR transiently pass through D, which is the only state that is less stable than the resting state. The reaction diagram shows that the desensitization step, A₂O  A₂D, and the recovery step, D  C, are each accompanied by a net stabilization of the system even though they are functionally inverse processes.

View larger version (15K): [in this window] [in a new window]   Fig. 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 GO values (kBT ) and were calculated from the ratios of the rate constants shown in Model IV. The sign of the GO 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.

Mechanistic Models

The term "desensitization" is a phenomenological one and does not imply any particular physical mechanism for AChR inactivation. In kinetic models of AChR operation, the classification of a state as being `C' or `O' relates to the conductance status of the pore, which in turn reflects the main allosteric transition of the protein. However, the classification of `D' makes no particular physical reference. In this section, we interpret the kinetic results using specific physical models for AChR desensitization.

Activation of AChR is a global change in the structure of the protein that includes rotation of helices at the transmitter binding sites and movement of residues in the pore domain (Unwin, 1995). The functional correlates of this event are a substantial decrease in the dissociation rate of ACh from the transmitter binding sites and a change from a nonconducting to a conducting pore. It is possible that desensitization reflects another such global change in the structure of the protein. Accordingly, the k^C_±D and k^O_±D rate constants of Model IV describe the rate constants for this additional, global transition.

If there is only one gate, desensitization can be thought of as a change in the coupling between the binding sites and the pore because the solitary gate closes without an accompanying increase in the dissociation rate constant of ACh. If this were so, the kinetic results indicate such interruption occurs readily only when the gate is open (A₂O  A₂D), but is reestablished readily only when the binding sites are empty (D  C). Moreover, the results indicate that desensitization is an energetically favorable transition when the sites are liganded, but is unfavorable when they are empty.

Certain evidence supports the allosteric hypothesis for AChR desensitization. High resolution electron microscopy reveals only a single structural element in the AChR channel that might serve as a gate (Unwin, 1993), although this barrier has not been detected by cysteine-scanning mutagenesis (Akabas et al., 1994). In addition, low resolution images of profoundly desensitized Torpedo AChR show a change in the tangential tilt of the extracellular domain of the  subunit, suggesting that a desensitization is accompanied by a large-scale change in the AChR structure (Unwin et al., 1988).

The observation that activated AChR desensitize much faster than resting AChR recalls the two-gate, "ball and chain" inactivation mechanism of some voltage-gated channels (Hoshi et al., 1991), in which an inactivation gate (i.e., a tethered blocking particle, or ball) prevents ion permeation presumably by interacting with residues in the pore that become exposed when the activation gate (which is coupled to the voltage sensor) is open. The simplest form of such a two-gate inactivation mechanism cannot pertain to AChR because neither the re