Introduction

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The family of neurotransmitter-gated ion channels is responsible for fast synaptic transmission in the peripheral and central nervous systems (CNS) (Barnard et al., 1987; Unwin, 1989; Betz, 1990). Functional regulation of these receptors by second-messenger systems has often been examined with respect to their involvement in neuronal plasticity (Swope et al., 1992; Smart, 1997). How receptor modulation contributes to possible dysfunction of receptors in disease states is less well understood. It is possible that various intracellular mechanisms required to regulate receptors under normal circumstances confer long-lasting changes during abnormal conditions. Such receptor regulation could underlie the "functional inactivation" of neuronal nicotinic acetylcholine receptors (nAChRs) that occurs during and after chronic exposure to nicotine (Lukas, 1991; Peng et al., 1994).

Neuronal nAChRs are amenable to a variety of physiologically relevant forms of regulation. The number and/or function of receptors can be increased through both cAMP-dependent and -independent mechanisms (Margiotta et al., 1987; Gurantz et al., 1993), by protein kinase C (PKC) (Downing and Role, 1987), by the neurotransmitters vasoactive intestinal peptide (Gurantz et al., 1994; Cuevas and Adams, 1996) and Substance P (Role, 1984), by changes in the extracellular concentration of Ca²⁺ (Mulle et al., 1992b; Vernino et al., 1992; Galzi et al., 1996) and through interactions with the cytoskeleton (Bencherif and Lukas, 1993). In addition, the permeation of Ca²⁺ through nAChR channels (Vernino et al., 1992) could activate intracellular cascades or other ion channels (Mulle et al., 1992a) and potentially induce changes in the phosphorylation states of specific nAChR subunits (Vijayaraghavan et al., 1990; Nakayama et al., 1993; Moss et al., 1996). The long-term functional consequences of such post-translational modifications remain largely unexplored.

Because of nAChR subunit diversity (McGehee and Role, 1995; Colquhoun and Patrick, 1997), their biochemical regulation may occur in a subtype-specific manner. Although it is unclear which subtypes of nAChRs predominate in CNS function, receptors containing 4 and 2 subunits contribute to a majority of the high-affinity nicotine binding sites in the brain (Whiting and Lindstrom, 1988; Flores et al., 1992). Furthermore, nicotine at levels related to tobacco use (Benowitz et al., 1989) both activates and desensitizes 42 nAChRs (Hsu et al., 1995; Fenster et al., 1997); desensitization may initiate the up-regulation of nAChR number that occurs during chronic nicotine exposure (Wonnacott, 1990; Schwartz and Kellar, 1985). Therefore, factors that regulate 42 nAChR function, especially those that modulate desensitization, may contribute to the long-term effects of nicotine on nAChR number and function. In this study, we investigate second-messenger modulation of 42 nAChRs expressed in Xenopus laevis oocytes. We show that the predominant role of Ca²⁺ and PKC is to regulate the rate of recovery from desensitization.

	Experimental Procedures

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Expression of Functional nAChRs in X. laevis Oocytes. Detailed procedures for preparation of oocytes have been described elsewhere (Quick and Lester, 1994). Briefly, oocytes were defolliculated and maintained at 18°C in incubation medium containing ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES, pH 7.4), 50 mg/ml gentamicin, and 5% horse serum. Subunit cRNAs were synthesized in vitro (machine message; Ambion, Austin, TX) from linearized plasmid templates of rat cDNA clones. A mutant 4 subunit (4^S336A) was created in which a consensus PKC phosphorylation site (serine 336) was mutated to alanine (pALTER 1; Promega, Madison, WI). The mutation was verified by sequencing. Oocytes were injected with between 5 and 25 ng/subunit/oocyte;  and  subunits were injected in 1:1 ratios.

Electrophysiological Recording. Whole cell currents were measured at room temperature (20-25°C), 24 to 96 h after RNA injection, with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA) in a standard two-microelectrode, voltage-clamp configuration. Electrodes were filled with 3 M KCl and had resistances of 0.5 to 2 M. Oocytes were clamped between 40 and 65 mV and superfused continuously in media containing 1.8 mM Ca²⁺ (control condition). In some experiments, 1.8 mM Ba²⁺ was substituted for Ca²⁺. In some of the early experiments, membrane- permeable drugs were applied by extracellular incubation. In the majority of experiments, drugs (25 nl) were injected into the oocytes 10 to 30 min before experimentation, so as to avoid direct extracellular effects of protein kinase activators/inhibitors on nAChR channels (see Reuhl et al., 1992). The approximate final concentrations were: phorbol-12-myristate-13-acetate (PMA), 200 nM to 2 µM; cyclosporin A, 500 nM to 1 µM; and calphostin C, 200 nM. Control incubations or injections were performed with vehicle solutions: 0.125% dimethyl sulfoxide for calphostin C and PMA treatments. Agonist-containing solutions were gravity-fed via a six-way manual valve (Rainin Instruments, Woburn, MA) to the oocyte in the recording chamber. Solution exchange considerations are discussed in Fenster et al. (1997). All salts and drugs were obtained from Sigma (St. Louis, MO). All currents were recorded on a chart recorder and on an 80486-based computer with AxoScope software (Axon Instruments) after 50- to 100-Hz low-pass filtering at a digitization frequency of 200 Hz. For slowly desensitizing responses, peak currents were assessed on-line from the digital readout of the amplifier.

Criteria for Data Selection. For accurate voltage-clamp, and to limit the activation of the endogenous Ca²⁺-activated Cl current in oocytes, nicotine-induced currents greater than 3 µA were not included in the data analysis; initial current amplitudes less than 50 nA were also excluded. Additionally, responses were at least 2-fold greater than the holding current, and the holding current at a given membrane potential was less than 100 nA. These criteria applied to all nAChR currents activated at the EC₅₀ concentration. Various desensitization parameters were estimated as described previously (Fenster et al., 1997). For the majority of experiments, desensitization was studied using the methods of Katz and Thesleff (1957) and Feltz and Trautmann (1982). Briefly, the fraction of activatable receptors before, during, and after a 30-min exposure to 300 nM nicotine was assessed from the amplitude of repetitively applied test pulses (5 s; 10-20 µM nicotine; interpulse interval, 5 min). The respective time courses of desensitization onset and recovery were estimated from exponential fits to the test pulse amplitude during and after the 300 nM nicotine application. Fits were sometimes constrained so that steady-state desensitization could not be less than zero. For means of comparison across different conditions, the magnitude of desensitization was calculated as the ratio (I_con I_final)/I_con, where I_con is the control test pulse amplitude and I_final is its amplitude at the end of the 30-min nicotine application. In a few experiments, desensitization onset and magnitude were determined from the response to a 2- to 3-min application of 10 µM nicotine. For statistical comparison of mean data, weighted-means Student's t tests (for unpaired comparisons) and paired Student's t tests (for paired comparisons) were performed. Comparisons of exponential fits were by nonlinear regression analysis using SPSS software (SPSS for Windows, Rel. 8.0.0. 1997; SPSS, Inc., Chicago, IL). All data are expressed as the mean ± S.E.M. Kinetic models of receptor desensitization were constructed using ScOP (Simulation Resources Inc., Berrien Springs, MI).

	Results

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Ca²⁺ Regulates Recovery from Desensitization. It has been shown previously that desensitization of 42 receptors induced by 3-min applications of 10 µM nicotine occurs in a biexponential manner, with similar time constants in the presence or absence of Ca²⁺ (Fenster et al., 1997). However, recovery from desensitization showed a marked dependence on the presence of extracellular Ca²⁺ (Fenster et al., 1997). To further investigate the role of Ca²⁺ on desensitization of 42 nAChRs, experiments were performed after extracellular Ca²⁺ was replaced by Ba²⁺ (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Recovery from desensitization is reduced in the presence of Ba2+. The time courses of desensitization and recovery were assessed from the inhibition of responses to a repetitively applied (5-min intervals) brief (5-10 s) test pulse of nicotine (10 µM) during incubations with 300 nM nicotine in the presence of extracellular Ca2+ (A) or Ba2+ (B). Time course plots of the test pulse amplitudes during 30 min applications of nicotine (left) and example responses from these experiments (right) are shown. Data are normalized to control nicotine responses before nicotine application. The solid lines show double exponential fits to the onset of desensitization, and the fast (f) and slow (s) time constants are indicated. Recovery from desensitization in the presence of Ca2+ was fit to both a single exponential (solid line) and a double exponential with a fast recovery time constant constrained to 2.5 min (dashed line). In the latter fit, the fast component represented 13% recovery and the slow recovery time constant was 54 min. Recovery from desensitization in the presence of Ba2+ was fit to a double exponential. The fast time constant represented 17% recovery. The slow time constant could not be defined.  in A (left) indicate the stability of the test pulse amplitude in the absence of continuous exposure to nicotine. The effects of a 10 min application of 300 nM nicotine on the rates of recovery from desensitization (thick lines) are shown for both Ca2+ (C; n = 4) and Ba2+ (D; n = 3). Recovery was well described by a single exponential component in Ca2+ but not in Ba2+. The fitted exponentials for onset and recovery for the 30-min applications are shown superimposed for comparison (thin lines).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   PKC activity regulates the rate of recovery from desensitization. The time courses of desensitization and recovery during a 30-min incubation with 300 nM nicotine in 42 expressing oocytes injected with PMA (A) or calphostin C (B). In each case, these oocytes were compared with control oocytes (uninjected/vehicle-injected) from the same oocyte batch. Time-course plots of the test pulse amplitudes (left) and example responses (right) are shown. Data are normalized to control nicotine responses before nicotine application. The solid lines show double exponential fits to the onset of desensitization with fast (f) and slow (s) time constants for A: control, 1.2 (63%) and 9 min (n = 4); PMA, 2.4 (59%) and 90 min (n = 7); and for B: control, 2.3 (64%) and 65 min (n = 4); calphostin C, 1.8 (82%) and 22 min (n = 4). In all cases, recovery from desensitization was well described by a single exponential and the time constant is indicated.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Phosphatase inhibition increases the rate of recovery from desensitization. The time courses of desensitization and recovery during a 30-min incubation with 300 nM nicotine in 42-expressing oocytes injected with cyclosporin A. These oocytes were compared with control oocytes (uninjected/vehicle-injected) from the same oocyte batch. Time-course plots of the test pulse amplitudes (A) and example responses (B) are shown. Data are normalized to control nicotine responses before nicotine application. The solid lines show double exponential fits to the onset of desensitization with fast (f) and slow (s) time constants for control of 2.7 (88%) and 22 min (n = 3) and for cyclosporin A of 2.2 (76%) and 11 min (n = 3). The time courses of recovery from desensitization were in both cases well described by a single exponential and the time constant is indicated.

PKC Regulates 42 Receptor Desensitization. Several mechanisms could account for the enhancement of recovery from desensitization by Ca²⁺. Two possibilities are that, in addition to enhancing activation (Mulle et al., 1992b; Vernino et al., 1992; Galzi et al., 1996), Ca²⁺ binding to an external site on 42 nAChRs also alters desensitization or that Ca²⁺ entry into the oocyte regulates the desensitization recovery process. To address the possible intracellular consequences of Ca²⁺, we assessed the effects of inhibition and activation of Ca²⁺-dependent protein kinases and phosphatases. For both muscle-type nAChRs (Hardwick and Parsons, 1996) and neuronal nAChRs in chromaffin cells (Khiroug et al., 1998), recovery from desensitization is dependent upon the state of phosphorylation and is controlled via a Ca²⁺-dependent phosphatase and PKC. Similar regulation of 42 nAChRs is possible, because 42 receptors are potential targets for modification by PKC (Goldman et al., 1987; Deneris et al., 1988). Injection of 42-expressing oocytes with PMA had two major effects on desensitization (Fig. 2A): compared with untreated oocytes from the same batch of cells, in the presence of extracellular Ca²⁺, there was an increase in the rate of recovery from desensitization (_rec = 14 min; n = 6; P < .05) and there was a reduction in the magnitude of desensitization (0.71; n = 10; P < .05). In another batch of oocytes, inhibition of PKC by calphostin C had the opposite effect (Fig. 2B). There was an increase in the magnitude of desensitization (0.92; n = 3; P < .05) and the rate of recovery from desensitization was slowed (_rec = 48 min; n = 3; P < .05). These data imply that PKC activity modulates 42 receptor desensitization: activation of PKC enhances recovery from desensitization and its inhibition reduces recovery.

Phosphatase Inhibition Enhances Rate of Recovery from Desensitization. The PMA and calphostin C data are consistent with the hypothesis that factors that promote phosphorylation enhance the rate of recovery from desensitization. An alternative method of promoting phosphorylation is to suppress dephosphorylation through phosphatase inhibition. Because the function of many ligand-gated channels is affected by the Ca²⁺-dependent phosphatase calcineurin (Yakel, 1997), the effects of the phosphatase inhibitor cyclosporin A on recovery from desensitization of 42 nAChRs were examined. Compared with a control group of oocytes from the same batch, the action of cyclosporin A was almost identical with the effects of activation of PMA (Fig. 3); that is, a dramatic increase in the rate of recovery from desensitization (_rec = 8 min; n = 3; P < .05) and a slight decrease in the magnitude of desensitization (0.62; n = 3; P > .05). These data support the suggestion that both the extent of 42 receptor desensitization and the rate of recovery are determined by the balance of phosphatase and kinase activity.

Elimination of a PKC Phosphorylation Site in 4 Subunit Inhibits Recovery from Desensitization. The data above imply that modulation of recovery from desensitization of 42-containing nAChRs may involve the Ca²⁺-dependent activation of PKC. One pathway for generating such specificity would be direct phosphorylation of the 4 or 2 subunit by PKC. In addition to two protein kinase A (PKA) sites, the 4 nAChR contains five consensus sites for PKC phosphorylation on the cytoplasmic loop between transmembrane regions 3 and 4, and the 2 subunit contains one PKC site in this region (Goldman et al., 1987; Deneris et al., 1988). One of these sites on the 4 subunit, serine 336, is analogous to a site (serine 333) on the muscle  subunit that is a likely site of PKC-dependent phosphorylation (Huganir et al., 1984). To test the hypothesis that this site is important for recovery from desensitization, a mutant 4 receptor subunit was created in which serine 336 was replaced with alanine. This subunit, denoted 4^S336A, was expressed in oocytes along with a wild-type 2 subunit. These mutant receptors formed ion channels that were activated and desensitized by nicotine (10 µM) in a manner similar to wild-type 42 nAChRs (Fig. 4). Dose-response relationships in the presence of Ca²⁺ yielded an EC₅₀ value of 13 µM, also similar to wild-type 42 receptors (Fenster et al., 1997). The onset of desensitization (nicotine 10 µM; 2 min) could be described by a single (1/6 cells) or a biexponential decay (5/6 cells), with fast [_f = 6.9 ± 1.6 s (24%)] and slow (_s = 135 ± 20 s) time constants; these data are not significantly different from wild-type receptors [_f = 5.1 ± 0.4 s (24%)]; _s = 109 ± 22 s). The magnitude of desensitization in 4^S336A2 nAChRs, estimated at the end of a 2-min nicotine application, was less than in wild-type 42 receptors: 0.46 ± 0.02 (n = 6) compared with 0.59 ± 0.02 (n = 7; P < .05).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Properties of mutant 4S336A2 receptors. A, concentration-response relationship for activation of mutant 4S336A2 receptors. The solid curve is a logistic fit to the data, with a Hill slope of 1.0. B, comparison of currents induced by 2-min applications of nicotine in wild-type 42- (left) and 4S336A2- (right) expressing oocytes.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Recovery from desensitization is reduced in mutant 4S336A2 receptors. A, the time courses of desensitization and recovery were assessed during a 30-min incubation with 300 nM nicotine in wild-type 42 (filled symbols) or mutant 4S336A2 (open symbols) receptors. Time-course plots of the test pulse amplitudes (left) and example responses (right) are shown. All oocytes are from the same batch. Data are normalized to control nicotine responses before nicotine application. The solid lines show double exponential fits to the onset of desensitization and the fast and slow time constants for wild-type 42 and mutant 4S336A2 receptors. Recovery from desensitization of wild-type 42 receptors has been fit to a single exponential with its time constant shown. Recovery from desensitization of mutant 4S336A2 receptors was best fit with the sum of two exponentials. B, Initial stability of peak amplitudes of test pulses. Brief pulses of nicotine (10 µM) were applied at 5- and 10-min intervals to oocytes expressing either wild-type 42 (filled symbols; n = 10) or mutant 4S336A2 (open symbols; n = 11). Time-course plots of the test pulse amplitudes (left) and example responses (right) are shown.

Models of 42 Receptor Desensitization. The most convenient method for understanding how phosphorylation could regulate 42 nAChR function is to examine the consequences of changing transition rates between the various distinct states in a Markov model. As discussed by others, cyclical schemes that incorporate desensitized conformations with high affinities for agonist can describe reasonably well both the onset and recovery of desensitization (Katz and Thesleff, 1957). The biexponential time course kinetics observed in the present study are most readily explained by a model with two desensitized states (Feltz and Trautmann, 1982; Boyd, 1987):

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View larger version (35K): [in this window] [in a new window]   Fig. 6.   Simulation of 42 receptor desensitization. A, initial stability of test pulse amplitude. Simulated responses to 10-s applications of nicotine (10 µM) at 10-min (left) or 5-min (right) intervals. The percentage loss of response is shown in each case after 20 min of stimulation. B, simulated desensitization experiment using 2-s pulses of nicotine (10 µM) applied at 5-min intervals before, during, and after exposure to 300 nM nicotine for 30 min. Desensitization onset and recovery are described by double exponentials (dashed lines) with time constants (and relative amplitudes) indicated. Test pulse responses were allowed to stabilize before simulated exposure of the model to 300 nM nicotine. C, time course of the change in the fraction of activatable receptors during exposure to various concentrations of nicotine. D, plot of the available receptor fraction after 20-min nicotine exposure (arrow in C) with respect to nicotine concentration. The sold line is a logistic fit to the simulated data.

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View larger version (24K): [in this window] [in a new window]   Fig. 7.   The effects of rate-constant changes on the time course of desensitization. A, simulated test pulse (nicotine, 2 s; 10 µM) amplitudes during and after a 30-min simulated exposure to 300 nM nicotine. The rate of recovery from desensitization is slowed by a 3-fold reduction in l2 (d2 was reduced by a similar amount to maintain microscopic reversibility). A 3-fold increase in l2 (and d2) increased the rate of recovery (B), whereas a 3-fold decrease in d+2 (and l+2) increased the fraction of receptors that recovered with a fast time constant and reduced the magnitude of desensitization (C). Opposite rate changes mimic the effects of PKC activation (D) and inhibition (E) (see Fig. 2). D, a 2-fold increase in l2 balanced by a 2-fold decrease in d+2 enhanced the rate of recovery and decreased the magnitude of desensitization. E, a 2-fold decrease in l2 balanced by a 2-fold increase in d+2 reduced the rate of recovery and increased the magnitude of desensitization. Open symbols indicate the test pulse amplitudes and solid lines are exponential fits. In A-E, the control behavior is shown with dashed lines. F, simulated currents in response to brief pulses of nicotine (3 min; 10 µM). From left to right, traces show responses with control rate constants and adjusted rate constants as in part E (calphostin C) and A (mutant). The fast desensitization time constant and its relative percentage are the same for each of the three simulations.

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	Discussion

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Protein phosphorylation is essential for G protein-coupled receptor desensitization (Freedman and Lefkowitz, 1996). In the case of ligand-gated channels, phosphorylation plays a modulatory rather than a necessary role in receptor desensitization (Huganir and Greengard, 1990). Examination of the functional properties of 42 nAChRs expressed in X laevis oocytes has revealed that recovery from desensitization is specifically amenable to certain forms of biochemical regulation. It is proposed that Ca²⁺ and factors that promote phosphorylation, possibly directly of the 4 subunit, enhance the overall rate of recovery from desensitization.

Desensitization of 42 Receptors. Since the initial studies of Katz and Thesleff (1957), much effort has been invested in defining the process of desensitization in both molecular and biophysical terms. Current models of desensitization for most ligand-gated ion channels are based around cyclical schemes with two distinct desensitized states (Sakmann et al., 1980; Feltz and Trautmann, 1982; Boyd, 1987). Consistent with these studies, we have shown that the time courses of desensitization onset and recovery display biexponential kinetics. It is possible that this type of behavior can be explained by the existence of two separate channels: one with fast desensitization properties and one with slow desensitization properties (for example, see Maconochie and Knight, 1992). However, after short applications of nicotine (10 min), the fast component dominates both the onset of and recovery from desensitization, whereas after 30-min exposure to nicotine, the slow component (80%) predominates during recovery. This implies that the fast and slow phases are not independent. These data are most readily explained by the existence of a single receptor type with complex desensitization characteristics (Feltz and Trautmann, 1982). After brief desensitization, most receptors have time only to reach a fast desensitized state from which recovery is also fast. During prolonged desensitization, more receptors are converted to a slowly reached desensitized state, from which recovery is also slow.

Regulation of Recovery from Desensitization. The involvement of phosphorylation in recovery from desensitization of neuronal nAChRs has recently been demonstrated (Khiroug et al., 1998). Boyd (1987), however, first suggested that a biochemical process may specifically regulate the slow phase of neuronal nAChR desensitization. In the present study, we have confirmed Boyd's idea. We find that Ca²⁺ and PKC are involved in the regulation of the slow phase of recovery from desensitization. For example, with Ba²⁺ substitution, 42 receptors displayed a marked reduction in the slow rate of recovery, with no other differences from control conditions. Mechanistically speaking, this can only be explained by a reduction in the rate constant that allows escape from the "deep" desensitized state D₂  D₁. The slower rate of recovery from desensitization in Ba²⁺-containing media implies that Ca²⁺ may be important for normal 42 receptor function. It seems unlikely that the action of Ca²⁺ results from direct binding to an extracellular site on the receptor (Mulle et al., 1992b; Vernino et al., 1992; Galzi et al., 1996) because Ba²⁺, which can replace Ca²⁺ in its ability to increase nAChR responses (Mulle et al., 1992b), does not substitute for Ca²⁺ in the enhancement of recovery from desensitization. Therefore, Ca²⁺ influx, perhaps in part via the nAChR channel itself (Mulle et al., 1992a; Vernino et al., 1992), may be important for its effects on recovery. Ca²⁺ could be acting directly on the intracellular face of the receptor (Miledi, 1980; Cachelin and Colquhoun, 1989) or through activation of various Ca²⁺-dependent kinases and phosphatases, as suggested for nAChRs in chromaffin cells (Khiroug et al., 1998). We have shown that activation and inhibition of PKC enhances and attenuates the slow rate of recovery from desensitization of 42 nAChRs, respectively, consistent with the idea that factors that promote phosphorylation facilitate recovery from the "deep" desensitized state. Because recovery from desensitization can be enhanced both by phosphatase inhibition and by PKC activation, it is suggested that the rate of recovery from desensitization will be governed by the relative balance of kinase and phosphatase activity. Because the effects of Ba²⁺ in wild-type 42 receptors are almost identical with the behavior of the mutant 4^S336A2 nAChR, we would argue that the dominant role of Ca²⁺ under the present conditions is to facilitate recovery from desensitization, possibly through activation of PKC.

4 Subunits are Potential Substrates for PKC. Direct phosphorylation of 4 nAChR subunits by PKA (Nakayama et al., 1993) is enhanced by chronic treatment with nicotine (Hsu et al., 1997). Although various muscle nAChR subunits act as PKC substrates (Huganir and Greengard, 1990), no equivalent direct PKC-mediated phosphorylation of neuronal nAChRs has been demonstrated. As predicted from our results with inhibition and activation of PKC, the PKC-site mutant 4^S336A2 nAChR exhibited a decrease in the slow time constant of recovery from desensitization, which suggests that PKC-dependent phosphorylation at this site may be important for recovery of function. However, the effects of inhibition of PKC were not restricted entirely to the recovery from desensitization; there was also an increase in the magnitude of desensitization. Based on our model, we suggest that this may be explained by an increase in the rate constant governing entry into the "deep" desensitized state. In other reports, the onsets of desensitization of muscle-type nAChRs (Huganir et al., 1986; Hoffman et al., 1994) and nAChRs in sympathetic ganglia (Downing and Role, 1987) are enhanced by PKA and PKC activation, respectively. In the case of chromaffin cells, any action of PKC on desensitization onset and/or steady-state response may have been missed (Khiroug et al., 1998), because the high concentrations of agonist used would rapidly drive the majority of receptors into the "shallow" desensitized state and the transition to the "deep" state would become largely silent (see Fig. 7F). Furthermore, because the rate of recovery from desensitization in the mutant receptor could be partially enhanced by a combination of cyclosporin A and PMA, it is likely that the effects of phosphorylation on desensitization are not limited to one site on 4 subunits. Indeed the 4 subunit alone contains five potential PKC sites (Goldman et al., 1987) and can be heavily phosphorylated in oocytes in vivo (Viseshakul et al., 1998). Our results do not necessarily indicate that PKC directly phosphorylates this site; rather, the mutation could be affecting the interaction of the 4 subunit with some intermediate protein or it could confer a conformational change in the subunit that itself alters the rate of recovery from desensitization. In this respect, we did observe a slight decrease in the magnitude of desensitization in mutant nAChRs that was not predicted.

Implications for Nicotine Addiction. Prolonged exposure to levels of nicotine related to use of tobacco up-regulates the number of high-affinity (42) nicotine binding sites in the CNS (Marks et al., 1983; Schwartz and Kellar, 1985; Flores et al., 1992) and in heterologous expression systems (Peng et al., 1994; Gopalakrishnan et al., 1996). In contrast to the increase in receptor number, the functional responsiveness of nAChRs is markedly reduced (Lukas, 1991; Marks et al., 1993; Peng et al., 1994). It has been suggested that reduced function is a consequence of nAChRs entering a "permanently inactive" state (Lukas, 1991; Peng et al., 1994), probably via agonist-induced desensitized conformations (Boyd, 1987). Previously we have demonstrated that 42 nAChRs have an intrinsically slow rate of recovery from desensitization after a 30-to 60-min treatment with levels of nicotine related to use of tobacco (Fenster et al., 1997). We suggest here that desensitization of nAChRs induced by prolonged exposure to nicotine may result in a reduced Ca²⁺ influx, thereby promoting the dephosphorylated state of 42 receptors. Recovery from the "deep" desensitized conformation would be markedly slowed, and receptors would become "trapped" in a chronically desensitized/deactivated state (Lukas, 1991; Peng et al., 1994). Indeed, it has been reported that prolonged treatment with PKC inhibitors will also drive 42 nAChRs to a functionally inactive conformation (Eilers et al., 1997). Chronic PMA treatment, which down-regulates PKC activity (Favaron et al., 1990), promotes an increase in the number of 42 receptors (Gopalakrishnan et al., 1997), consistent with the suggestion that the dephosphorylated state of the receptor could either directly or indirectly serve as a signal for preventing receptor turnover (Peng et al., 1994).