Acute Activation, Desensitization and Smoldering Activation of Human Acetylcholine Receptors

The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained “smoldering activation” occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)2β2 and (α4β2)2α4. The (α4β2)2β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)2α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.


Introduction
The component of tobacco that drives its compulsive use is nicotine, an alkaloid that acts on nicotinic acetylcholine receptors (AChRs) in the brain. AChRs are a heterogeneous family of ligand-gated cation channels which consist of five homologous subunits arranged around a central pore [1,2]. They are heteropentamers formed of combinations of a and b subunits, or homopentamers formed entirely of a7 subunits [3]. Heteromeric AChRs usually have two ACh binding sites that are formed between adjacent a and b subunits. The remaining subunit is in the ''accessory'' position. While the accessory subunit does not usually form part of a binding site for ACh, it has major effects on responses to nicotinic agonists, antagonists and allosteric modulators. There is a third ACh binding site in the (a4b2) 2 a4 stoichiometry, formed at the interface between the a4 accessory subunit and another a4 subunit [4,5].
The predominant AChR subtypes in human brain are heteromeric AChRs comprised of combinations of a4 and b2 subunits, alone or in combination with other subunits, such as b3, a5 or a6, or homomeric AChRs comprised of a7 subunits. Selfadministration of nicotine is inhibited by knockout of a4, b2, or a6 subunits [6], but is increased by knockout of a5 subunits [7]. AChRs containing the a3 subunit are the predominant postsynaptic AChRs in the autonomic and enteric nervous systems [8]. In the brain, a3 and b4 subunits are prominent only in the medial habenula [9].
Although nicotine is a drug of abuse, it also has many positive effects that could be exploited therapeutically. In addition to their use for treating nicotine addiction, nicotinic agonists are being developed for treatment of disorders ranging from Alzheimer's disease to schizophrenia [10]. Varenicline and cytisine have been promoted for treating nicotine addiction because they are high affinity partial agonists that displace nicotine and decrease its rewarding effects by attenuating dopamine release in the mesolimbic system [11]. However, many of the clinical effects of nicotine and partial agonists are mediated by desensitization of AChRs. Sazetidine-A is a very potent and high affinity acute agonist, and a very efficient desensitizer. It produces many of the in vivo effects of nicotine and partial agonists such as inhibition of nicotine self-administration, increased attention, pain relief, reduction in anxiety and depression, and weight reduction, suggesting that these effects may result more from desensitization than from activation [12][13][14][15][16][17].
All of these cholinergic ligands upregulate (a4b2) 2 b2 AChRs in tissue culture, and all but sazetidine-A also do so in vivo [18]. Sazetidine-A may have sufficient membrane permeability in vivo to desensitize AChRs on neuron surfaces, but insufficient to achieve intracellular concentrations sufficient to promote assembly of (a4b2) 2 b2 AChRs [18,19]. Transient application of nicotine or other agonists activates AChRs, opening the cation channel resulting in depolarization and other effects mediated by entry of cations, followed by acute desensitization and then rapid recovery. Chronic exposure to agonists causes prolonged desensitization. In typical physiological assays, the effects of these drugs are observed over seconds or minutes. However, in vivo they are present over many hours. ''Smoldering activation'' occurs at agonist concentrations at which some AChRs are desensitized while others are activated. This can occur within minutes after recovery from acute activation or after prolonged continuous exposure to agonists. The relative contributions of acute activation and chronic desensitization of AChRs in causing as well as treating nicotine addiction, or mediating the beneficial effects of nicotine are being actively investigated [18,20].
We have established a number of transfected cell lines that permanently express human AChR subtypes [21][22][23][24][25]. The a4b2 expressing cell line contains a mixture of two stoichiometries differing by the presence of a4 or b2 in the accessory position [22]. A similar mixture of stoichiometries is found in brain [26,27]. The (a4b2) 2 b2 stoichiometry is more sensitive to activation and upregulation by nicotine, desensitizes more slowly and is less permeable to calcium [22,28]. It is sensitive to activation by sazetidine-A, but not varenicline or cytisine, whereas the (a4b2) 2 a4 stoichiometry is sensitive to activation by varenicline and cytisine but not sazetidine-A [29,30].
It is difficult to measure chronic desensitization of human AChRs using classical electrophysiological techniques such as human AChRs expressed in Xenopus oocytes or patch clamp studies on individual cells. Furthermore, until recently, it has not been possible to express sufficient amounts of a7 AChRs in human cell lines for functional assays. To obtain sufficient expression of a7 AChRs, we used chemical chaperones to promote assembly of human a7 in a cell line which co-expresses a7 and the AChR chaperone protein RIC-3 [25].
In this study, we examined the effects of acute and prolonged application of nicotine and three drugs which are known to inhibit nicotine self-administration (varenicline, cytisine and sazetidine-A), on human a4b2, a3b4 and a7 AChRs. These studies confirm and extend basic expectations of the properties of these agonists. The agonists fully desensitized these AChR subtypes with the exception of sazetidine-A on (a4b2) 2 a4. We have defined the range of concentrations of each of these agonists which can sustain smoldering activation of these AChRs. For a4b2, but not a3b4 or a7 AChRs, smoldering activation occurs at concentrations of nicotine that are sustained in smokers.

Cell Lines and Transfection
The parental cell line used for transfections was tsA201, derived from the human embryonic kidney cell line (HEK) 293 [33]. The cell line permanently expressing a4b2 AChRs [22,23] has a mixture of two a4b2 stoichiometries, namely (a4b2) 2 a4, which has lower sensitivity, and (a4b2) 2 b2, which has higher sensitivity to activation by ACh [22]. The cell line permanently expressing a3b4 AChRs has been described [21,34]. A stable cell line expressing functional a7 AChRs was prepared by initially transfecting the tsA201 cell line with cDNA for a7 and subsequently transfecting with cDNA for the AChR-selective chaperone, human RIC-3. The expression of functional a7 AChRs in this cell line was further increased by growth in the chemical chaperones valproic acid (VPA, 1 mM) and 4-phenylbutyric acid (PBA, 3 mM) for at least 2 weeks before functional assays [25].
All transfected cell lines were grown in Dulbecco's modified Eagle's medium (InVitrogen, Carlsbad, CA) with 10% fetal bovine serum (Hyclone, Logan, UT) supplemented with 2 mM glutamine. The expression of a4, a3 and a7 was maintained with Zeocin (0.5 mg/ml) and the expression of b2, b4 and RIC-3 was maintained with G418 (0.6 mg/ml). The cell lines were grown at 37uC with 5% CO 2 in a humidified atmosphere.

Acute Activation
Responses to nicotinic agonists were determined using a FLEXStation microplate fluorometer (Molecular Devices, Sunnyvale, CA) as described [23]. For cell lines expressing a4b2 and a3b4 AChRs, the cells were plated at 10 5 cells/ml (100 ml/well) in black-walled clear-bottom 96 well plates (Costar, Fisher Scientific, Pittsbugh, PA), and incubated for 48 hours prior to assaying responses to various nicotinic agonists. The a7/RIC-3 expressing cells were plated at 5610 5 /ml in black-walled clear-bottom 96 well BioCoat plates (BD Biosciences, Franklin Lakes, NJ) in the continued presence of VPA and PBA, with the addition of 5% human AB serum (Pel-Freez Biologicals, Rogers, AR) to further increase expression of a7 [35]. The cells were then grown for 24 hours prior to the assay.
To measure responses to various nicotinic agonists, 100 ml of a fluorescent dye which is sensitive to changes in membrane potential (Molecular Devices) was added to the wells. The dye was prepared according to the manufacturer's instructions, with the addition of atropine (0.5 mM) to block muscarinic responses. The plates were then incubated at 37uC for one hour prior to the assay. Serial dilutions of agonists were then prepared in Hanks Balanced Salt Solution (HBSS) in V-shaped 96-well plates (Fisher Scientific Co, Pittsburgh, PA). Fluorescent responses were measured in the FLEX Station at 25uC, with an excitation wavelength of 530 nm and an emission wavelength of 565 nm. Various agonists (50 ml) were added after the first 20 seconds and responses were followed for 60-120 seconds.

Chronic Desensitization
To measure desensitization, plates were prepared as for agonist assays. Sixteen hours prior to the assay, serial dilutions of agonists, prepared in regular growth medium, were added to the plates. One hour before the assay, the membrane potential-sensitive fluorescent dye with 0.5 mM atropine was added to the wells. For a4b2 AChRs, responses were measured to 3 mM ACh (to detect responses of (a4b2) 2 b2 AChRs) and 100 mM ACh (to detect responses of both stoichiometries). For a3b4 and a7 AChRs, responses to saturating concentrations of ACh (1 mM for a3b4 and 10 mM for a7) were measured. Each data point represents the average of peak values for individual dose response curves from 4-8 wells.
Expression of (a4b2) 2 b2 AChRs To determine which stoichiometry of a4b2 AChRs was affected by sazetidine-A, we performed short term transfection of b2 subunits into the a4b2 expressing cell line. To increase the expression of the higher sensitivity (a4b2) 2 b2 stoichiometry, the cell line expressing a4 and b2 was cotransfected with human b2 (pRc-CMV/Geneticin) using the FuGene6 transfection agent (Roche Diagnostics, Indianapolis, IN). On the following day, 0.5 mM nicotine was added to further increase the expression of the (a4b2) 2 b2 stoichiometry. After incubation for 24 hr with nicotine, the cells were washed twice with growth medium, and then serial dilutions of sazetidine-A were added for 6 hours prior to the assay. Desensitization by sazetidine-A was measured using the FlexStation as described above, with 3 mM and 100 mM ACh.

Smoldering Activation following Acute Desensitization
Responses of a4b2, a3b4 and a7 AChRs to the acute application of ACh (100 mM), nicotine (16 mM), varenicline (4 mM), cytisine (16 mM) and sazetidine-A (62.5 nM) were measured using the FlexStation as described. These drug concentrations were selected because they gave maximum sustained responses to these agonists. Results were expressed as a percentage of maximum response to ACh. Responses were monitored for 10 minutes, and then specific antagonists were added and responses recorded for another two minutes. The antagonists were dihydroberythroidine (DHbE) (1 mM) for a4b2, mecamylamine (MCA (10 mM) for a3b4 and methyllycaconitine (MLA) (10 mM) for a7. These concentrations of antagonists were selected because they were sufficient to inhibit responses to the tested agonists without causing activation themselves.

Statistics
Data were graphed using GraphPad Prism software. Non-linear models (sigmoidal dose-response with variable slope or two site competition) were used to fit the concentration response curves. The EC50 and IC50 were calculated from the curves. Kaleidagraph software was used to determine Hill slopes and standard errors of the EC50 and IC50.

Acute Responses to Nicotinic Agonists
Acute responses of a4b2, a3b4 and a7 AChRs were tested after application of a range of concentrations of ACh, nicotine, varenicline, cytisine and sazetidine-A, using the FlexStation with an indicator which is sensitive to changes in membrane potential. Responses of these AChRs to saturating concentrations of ACh are shown in Figure 1. The kinetics of responses of the other agonists were very similar to those of ACh (data not shown). The effects of saturating concentrations of ACh on a4b2 and a3b4 AChRs had the same appearance, with a maximum response reached within 40 seconds of agonist application. The responses of a7 AChRs were quite different with a maximum response within 5 seconds, followed by acute desensitization. However, the rate of acute desensitization was less than detected with electrophysiological techniques [36][37][38]. The amplitudes of responses of saturating concentrations of ACh (expressed as relative fluorescence units) were similar for a4b2 (167,000+/218,000) and a3b4 (161,000+/ 214,000), but significantly lower for a7 AChRs (54,000+/23000), probably as a result of rapid desensitization [36].
The concentration response curves for various agonists on a4b2, a3b4 and a7 AChRs are shown in Figure 2. The EC50's are summarized in Table 1. Nicotine had an efficacy comparable to that of ACh on a4b2* AChRs, whereas varenicline, cytisine and sazetidine-A were partial agonists, with efficacies of 48% for varenicline, 34% for cytisine and 44% for sazetidine-A. All of the tested compounds were full agonists on a3b4 and a7 AChRs. The EC50 values for a7 were lower than often reported by electrophysiological techniques. The EC50 values for a7 vary widely according to the assay method [36,[39][40][41]. Millisecond agonist kinetics are probably irrelevant for drugs present in the body for hours. It is likely that sustained smoldering activation and sustained antagonism due to desensitization are the most relevant factors. The fluorescence indicator is a more sensitive measure of a7 activation, although the response kinetics are slower [38].
The cell line transfected with a4 and b2 subunits expresses a mixture of (a4b2) 2 b2 and (a4b2) 2 a4 stoichiometries, which have different sensitivities to nicotine and other agonists. The concentration response curves for acetylcholine, nicotine and varenicline fit with a two-site competition model, likely indicating that these agonists have different effects on the two stoichiometries. On the other hand, the dose response curves for cytisine and sazetidine-A were monophasic, likely because these agonists act on only one stoichiometry.
The EC50 of nicotine for the more sensitive (a4b2) 2 b2 stoichiometry was 0.18 mM, which is within the range of nicotine levels detected in smokers (see Discussion). For varenicline, the EC50 for the more sensitive stoichiometry was 0.20 mM, which is close to the peak blood levels of 0.1 mM detected in humans after a 1 mg dose of this drug [42]. On the other hand, the EC50 for nicotine on a3b4 was 9.7 mM and for a7 AChRs was 0.75 mM, levels which cannot be reached in the systemic circulation. The EC50 for varenicline on a7 AChRs was 0.4 mM, which is close to levels reached in humans after a dose of 1 mg [42]. The EC50 of cytisine for a4b2 AChRs was 5.5 mM. It is uncertain whether this is a clinically achievable level. Sazetidine-A was the most potent of all the agonists on a4b2 AChRs (EC50 = 0.0023 mM). In mice treated with 2 mg/kg sazetidine-A, serum levels of 1.6 mM and brain levels of 0.15 mM are reached [19].

Desensitization
To assess desensitization, cell lines expressing human AChRs were incubated overnight with a range of concentrations of agonists, and responses to ACh were then measured. For a4b2* AChRs, activity was assayed using 3 mM ACh (to assay function of the more sensitive (a4b2) 2 b2 stoichiometry), and 100 mM ACh (to assay function of both stoichiometries). For the other AChRs, saturating concentrations of ACh were applied (1.0 mM for a3b4 and 10 mM for a7). Responses for the three different AChRs are shown in Figures 3, 4, 5, 6, along with the dose response curves for activation (the same as shown in Figure 2). The range of concentrations at which both sustained activation and desensitization can occur (''smoldering activation'') was calculated by multiplying the acute response to agonists at each concentration by the fractional response remaining after desensitization.
The results for a4b2 are shown in Figure 3. The intercept of the activation and desensitization curves is shown in Table 1. For nicotine, the area of overlap of the activation and desensitization curves (using 3 mM ACh) was centered at 0.13 mM (summarized in Table 1). This is within the range of nicotine concentrations found in the blood of smokers (0.058-0.34 mM [43] ). The extent of calculated smoldering activation at a clinically relevant concentration of 0.1 mM nicotine was 6% of maximum response for the (a4b2) 2 b2 stoichiometry and 18% for the (a4b2) 2 a4 stoichiometry. Nicotine is capable of sustaining a calculated smoldering response of 54% of maximum response on the (a4b2) 2 a4 stoichiometry. However, this requires a nicotine concentration of 1.8 mM, which far exceeds concentrations sustained in smokers. Likewise, for varenicline, the area of overlap of the activation and desensitization curves (using 3 mM ACh) was centered at 0.16 mM. Levels of 0.4 mM are reached in patients on therapeutic doses of this drug [42]. Smoldering activation by varenicline or cytisine is likely mediated by the (a4b2) 2 a4 stoichiometry. For example, at a varenicline concentration of 1 mM, .90% of (a4b2) 2 b2 AChRs are desensitized, whereas ,50% of (a4b2) 2 a4 AChRs are desensitized. At 1 mM varenicline, the smoldering activation is 4.3% of the maximum response for (a4b2) 2 b2 AChRs compared to 23% for (a4b2) 2 a4 AChRs. Desensitization is more likely to account for the effectiveness of these agonists in smoking cessation than is activation, since knock out of a4 or b2 (but not a7) eliminates nicotine self-administration [6].
For cytisine the area of overlap of the activation and desensitization curves for a4b2 sites (with 3 mM ACh) was centered at 0.45 mM. When desensitization by cytisine was assayed with 100 mM ACh, there was a plateau on the dose response curve for cytisine concentrations above 10 nM.
For sazetidine-A the area of overlap of the activation and desensitization curves with 3 mM ACh centered around 0.0015 mM. There was also a plateau on the desensitization curve for a4b2 AChRs with sazetidine-A using 100 mM ACh, suggesting that sazetidine-A desensitized the response of the (a4b2) 2 b2 but not the (a4b2) 2 a4 stoichiometry. To resolve the contributions of the two stoichiometries to the effects of sazetidine-A, we transfected the a4b2* expressing cell line with additional b2 subunits, and then cultured the cells in the presence of 0.5 mM nicotine in order to express predominantly the (a4b2) 2 b2 stoichiometry. As shown in Figure 4, the desensitization curves for sazetidine-A, using both 3 mM and 100 mM ACh overlapped. These curves are very similar to the one shown in Figure 3 for sazetidine-A on mixed stoichiometries of a4b2 tested with 3 mM ACh (which activates predominantly the (a4b2) 2 b2 stoichiometry). This indicates that sazetidine-A desensitizes only the (a4b2) 2 b2 stoichiometry. The plateau on the dose response curve for 100 mM ACh with mixed stoichiometries of a4b2* likely indicates a lack of agonist and desensitizing activity of sazetidine-A on the less sensitive (a4b2) 2 a4 stoichiometry. Carbone et al. [30] reported that sazetidine-A is a full agonist at (a4b2) 2 b2 AChRs but had ,1% efficacy on the (a4b2) 2 a4 stoichiometry. Sazetidine-A may not be able to bind, activate or desensitize the third ACh binding site present at the a4/a4 interface in the (a4b2) 2 a4 stoichiometry [4,5]. Sazetidine-A has by far the highest affinity of these agonists at the a4b2 binding sites and is exceptionally potent at inhibiting nicotine self-administration in rats [12]. This implies that  inhibition of nicotine self-administration can be mediated by desensitizing a4b2* AChR responses through their a4b2 binding sites. The desensitizing effects of sazetidine-A are known to persist long after brief acute activation [44]. For all of the tested agonists, a3b4 AChRs were much less sensitive to both activation and desensitization than were a4b2 AChRs ( Figure 5). The areas of overlap for the nicotine and varenicline activation and desensitization curves correspond to much higher drug levels than can be achieved in humans.
As shown in Figure 6, the activation and desensitization curves for a7 AChRs were much steeper than for either a4b2 or a3b4, as expected since a7 AChRs have five ACh binding sites acting cooperatively to activate this AChR (rather than two for (a4b2) 2 b2 and a3b4, or three for (a4b2) 2 a4). For nicotine, the area of overlap of the activation and desensitization curves for a7 corresponds to concentrations of nicotine that are higher than can be reached in humans, with an intercept of the nicotine activation and desensitization curves of 1.7 mM. However, for varenicline, the area of overlap of the activation and desensitization curves for a7 corresponds to concentrations that are within a clinically achievable range, with the intercept of the curves at 0.4 mM.

Smoldering Activation Following Acute Desensitization
We evaluated the kinetics of responses over several minutes to concentrations of the various agonists that gave maximum sustained responses. The results were expressed as a percentage of the maximum response to ACh. As shown in Figure 7, for a4b2 and a3b4 AChRs, following acute activation and partial desensitization, there was a low level of sustained activation lasting at least 10 minutes. This sustained response was abrogated by the application of specific antagonists after 10 minutes (dihydroberythroidine (DHbE) (1 mM) for a4b2 or mecamylamine (MCA) (10 mM) for a3b4). For a7 AChRs, the initial activation and desensitization was more rapid than for a4b2 or a3b4 AChRs. The residual response after 10 minutes was abrogated by the application of the a7 antagonist methylycaconitine (MLA) (10 mM).
Thus, small but significant smoldering responses can be maintained for a period of minutes after acute activation. With a3b4 and a7 subtypes, these effects may not be significant at drug concentrations obtained in vivo. With a4b2 subtypes, small but significant effects may occur in vivo, and may contribute to nicotine addiction.

Discussion
In this study, we utilize human AChRs cloned in human cells to examine the dual effects of activation and desensitization by nicotinic agonists. These transfected cell lines have advantages over AChRs expressed in Xenopus oocytes, which can retain nicotine and other agonists, and release them slowly, making it difficult to assess desensitization [45]. This is not an issue with HEK cells, which are much smaller than oocytes and lack their internal yolk compartments or other reservoirs that may account for tertiary amine uptake. Our binding studies with nicotine and epibatidine to live AChR-expressing HEK cells show that unbound agonists are easily washed away, indicating that these cells to not retain tertiary amines (unpublished results). It has not previously been possible to measure function of a7, because levels of expression were too low for functional assays. We have overcome this limitation by co-transfection with RIC-3 and by the use of chemical chaperones [25]. Human cell lines expressing specific AChR subtypes can be used for screening and evaluating novel compounds with activity on these AChRs.
Although nicotine, varenicline, cytisine and sazetidine-A are all agonists, their behavioral effects may depend as much on desensitization as activation. Most in vitro studies of nicotinic agonists have examined the acute effects of these drugs over seconds to minutes. It is unclear how this relates to the in vivo setting, where the drugs are present for hours or days. If the major mechanism by which these partial agonists inhibit nicotine selfadministration is desensitization, then this clinical effect may depend primarily on their binding affinity, rather than EC50 or efficacy.
Cytisine is a plant alkaloid used predominantly in Europe as an aid for smoking cessation [46]. It is a partial agonist on a4b2 AChRs [47]. However, its clinical utility has been limited by poor absorption and limited brain penetration. Using cytisine as a lead compound, Coe et al. synthesized a series of a4b2 partial agonists. One of these compounds, varenicline, was selected for further development because of its improved potency and efficacy [11]. Clinically, varenicline has been shown to improve long-term smoking cessation rates compared to unassisted quit attempts or bupropion (see [48] for review). It is now widely used for smoking cessation.
Sazetidine-A is a novel nicotinic receptor ligand that is highly selective for a4b2 AChRs [44]. It has potential applications for treating nicotine addiction [12], as well as depression [15,16,49] calculated by multiplying the extent of acute activation by the extent of sustained desensitization at each concentration. For nicotine, the area of overlap for the more sensitive (a4b2) 2 b2 stoichiometry was centered at 0.13 mM, which is a concentration typically found in smokers. Likewise for varenicline, the area of overlap for the more sensitive (a4b2) 2 b2 stoichiometry was centered at 0.16 mM, which corresponds to peak concentrations achieved in humans. Sazetidine-A was highly potent at activating as well as desensitizing a4b2 AChRs. The area of overlap for (a4b2) 2 b2 AChRs was centered at 1.5 nM. When 100 mM ACh was used for desensitization, there was a plateau on the dose response curve beginning at around 10 nM. doi:10.1371/journal.pone.0079653.g003 Figure 4. Sazetidine-A desensitization of the more sensitive (a4b2) 2 b2 stoichiometry with 3 mM and 100 mM ACh. Cells stably expressing a4b2 AChRs were further transfected with b2 subunits and cultured in nicotine as described, to enrich for the sensitive (a4b2) 2 b2 stoichiometry. Responses were measured with the FlexStation using a membrane potential sensitive indicator, and results were expressed as a percentage of maximum fluorescence. The responses to both 3 mM and 100 mM ACh overlapped, likely indicating that only the (a4b2) 2 b2 stoichiometry contributes to desensitization. The plateau on the desensitization curve with sazetidine-A on mixed stoichiometries of a4b2* (shown in Figure 3) indicates that the (a4b2) 2 a4 stoichiometry is not desensitized even at high concentrations of sazetidine-A. doi:10.1371/journal.pone.0079653.g004 and pain [14,50]. Initially it was reported to desensitize a4b2 AChRs in the absence of activation, but did not appear to either activate or desensitize rat a3b4 AChRs [44]. However, subsequently Zwart et al. [51], using Xenopus oocytes expressing human a4 and b2 subunits, found that sazetidine-A was a potent agonist for both a4b2 stoichiometries. It was a full agonist on the (a4b2) 2 b2 stoichiometry but had only 6% activity on (a4b2) 2 a4. Using pentameric concatenated (a4b2) 2 b2 and (a4b2) 2 a4 AChRs expressed in Xenopus oocytes, Carbone et al. [30] found that sazetidine-A was a full agonist on (a4b2) 2 b2 but was a partial agonist with very low efficacy on (a4b2) 2 a4 AChRs.
We found that varenicline, cytisine and sazetidine-A are partial agonists on the mixture of a4b2 AChR subtypes, but full agonists on a3b4 and a7 AChRs. While varenicline and cytisine are partial agonists on a4b2 AChRs, they are capable of fully desensitizing these AChRs to the effects of ACh. On the other hand, for sazetidine-A, full desensitization was not reached even at high concentrations, presumably because this drug has no agonist activity on the a4/a4 ACh binding site of (a4b2) 2 a4 AChRs. Varenicline, cytisine and sazetidine-A also partially upregulate a4b2 AChRs relative to nicotine (data not shown).
Acute activation of AChRs occurs within seconds of application of the agonist and is followed by acute desensitization. In the continued presence of agonist over several minutes, there is a low level of residual activation, which can be blocked by the application of specific antagonists. In the presence of agonist over many hours, there is complete desensitization of all the tested AChR subtypes, with the exception of sazetidine-A on the (a4b2) 2 a4 stoichiometry.
We propose that the area of overlap of the activation and desensitization curves defines the range of concentrations over which smoldering activation can be sustained. For a4b2 AChRs, the range of smoldering activation for nicotine and varenicline corresponds to concentrations that can be achieved clinically. However, for nicotine, the range of smoldering activation for a3b4 and a7 AChRs exceeds concentrations that can be reached in humans. For varenicline the range of smoldering activation for a3b4 AChRs exceeds clinically achievable levels. However, for a7 the range of smoldering activation corresponds to drug levels that can be reached clinically. This suggests that varenicline may have a clinical effect on a7 AChRs, which could contribute to the undesirable side effects of this drug. The a4b2 cell line has a mixture of (a4b2) 2 b2 and (a4b2) 2 a4 stoichiometries. In order to distinguish the effects of the two stoichiometries on the desensitization of a4b2 AChRs, we used two concentrations of ACh, namely 3 mM (to detect effects on the high sensitivity stoichiometry), and 100 mM (to detect effects on both stoichiometries). Recent reports indicate that the low sensitivity (a4b2) 2 a4 stoichiometry has a third ACh binding site at the interface between adjacent a4 subunits, resulting in an intrinsic bimodal concentration response curve with an additional low sensitivity component to the response [4,5]. Because we did not examine pure populations of (a4b2) 2 a4 AChRs, we were unable to detect this.
As expected, the agonist effect of nicotine on a4b2 AChRs was bimodal. For the more sensitive stoichiometry of a4b2, the EC50 for activation (0.18 mM) and the intercept of the activation and desensitization curves (0.13 mM) correspond to levels of nicotine that are clinically relevant (see below). On the other hand, the EC50's and the intercepts of the activation and desensitization curves of nicotine for a3b4 and a7 AChRs are well above the range of concentrations that are achieved in smokers, indicating that these AChRs are unlikely to be involved with the reinforcing properties of nicotine. This is consistent with studies in transgenic mice showing that a7 does not contribute to nicotine selfadministration, whereas the a4 and b2 subunits are both necessary and sufficient to maintain nicotine self-administration [6].
Apart from our results on (a4b2) 2 b2 for desensitization by sazetidine-A, we only examined mixed stoichiometries of a4b2. The selection of 3 and 100 mM ACh for desensitization of a4b2 AChRs may not completely separate the effects of the high and low sensitivity stoichiometries. While the use of chimeric or concatameric AChRs may separate the effects of the different stoichiometries of a4b2 AChRs, cell lines with a mixture of stoichiometries may be more representative of in vivo effects.
In active smokers, the majority of a4b2 AChRs in the brain are saturated [52], and thus are likely in a desensitized state. There is no information in humans on levels of nicotine in the brain during active smoking, but they are likely to be significantly higher than blood levels. Peak nicotine concentrations in venous blood of heavy smokers vary from 9.4-55.1 ng/ml (0.058-0.34 nM) [43].
At the clinically relevant nicotine concentration of 0.1 mM, the extent of smoldering activation was higher for (a4b2) 2 a4 (18%) than for (a4b2) 2 b2 (6%). This indicates that the stoichiometry Figure 6. Activation and Desensitization of a7 AChRs by Various Agonists. Responses were measured using the FLEXStation with an indicator sensitive to changes in membrane potential. Results were expressed as a percentage of maximum fluorescence. Activity remaining after 16 hours desensitization by the indicated concentrations of agonist was assayed using 10 mM ACh. The extent of smoldering activation (shaded area) was calculated by multiplying the extent of acute activation by the extent of sustained desensitization at each concentration. For nicotine, the intercept of the activation and desensitization curves was 1.7 mM (well above the clinically achievable range). However, for varenicline, the intercept of the activation and desensitization curves was 0.4 mM, a concentration which can be reached with therapeutic doses of this drug. doi:10.1371/journal.pone.0079653.g006 which is less sensitive to acute activation is more sensitive to smoldering activation by nicotine. This may be due to the fact that the amplitude of response of the (a4b2) 2 a4 stoichiometry is 4-11 fold greater than that of (a4b2) 2 b2, probably as a result of greater probability of channel opening when three ACh binding sites are occupied [4,5].
Our results show that cytisine is a partial agonist on a4b2 and a full agonist on a3b4 and a7, confirming what others have found [53]. The EC50 for cytisine on a4b2 was 5.5 mM. It is uncertain whether this concentration is clinically relevant, as drug levels of cytisine that can be reached in humans are not yet defined [54]. Using concatameric as well as unlinked a4b2 AChRs, Carbone et al. found that cytisine was a partial agonist on (a4b2) 2 a4, but was inactive on (a4b2) 2 b2 [30].
Peak varenicline levels of 0.48 mM are reached after 14 days on a standard dose of 1 mg twice daily [55]. We found that the EC50 for the more sensitive stoichiometry of a4b2 was 0.2 mM, which is within the therapeutic range, accounting for the therapeutic efficacy of this drug in treating nicotine addiction. The EC50 for a7 was 0.4 mM, also within the therapeutic range. The effect of varenicline on a7 AChRs may account for some of the toxicity of this drug. Nausea, which is a dose-limiting toxicity of varenicline, probably results from activation of 5HT 3 receptors [56]. The cause of the rare psychotic effects of varenicline which have led to the black box warning are unclear.
We found that sazetidine-A was a partial agonist on a4b2 and a full agonist on a3b4 and a7 AChRs. It was highly selective for a4b2 AChRs, with an EC50 of 0.023 mM, compared with 0.17 mM for a3b4 and 1.2 mM for a7. However, using this fluorescence assay, we detected much greater activity of sazetidine-A on human a3b4 and a7 AChRs than did Liu et al. [57] with a rubidium efflux assay on rat a3b4 and a7 AChRs. Using transient transfection of b2 to the a4b2 expressing cell line we were able to resolve the effects of sazetidine-A on the two a4b2 stoichiometries. We found that sazetidine-A desensitizes only the (a4b2) 2 b2 stoichiometry. The differential effect on the two stoichiometries may explain the discrepant reports in the literature regarding whether or not sazetidine-A can activate a4b2 AChRs.
The results reported here allow us to speculate on the effects of prolonged presence of these agonists on endogenous cholinergic signaling in vivo as well as modulation of the effects of nicotine in smokers. Since varenicline is a partial agonist with greater affinity than nicotine and consequently more potency at desensitizing, the smoldering activation produced by nicotine on a4b2 AChRs will be reduced in the presence of varenicline. The net effect of varenicline will be antagonistic to both the effects of nicotine and endogenous ACh signaling.
Cytisine has lower efficacy than varenicline on a4b2 but also has lower affinity and consequently less potency at desensitizing. The net effect of cytisine will be antagonistic to both the effects of nicotine and endogenous ACh and it will decrease smoldering activation by nicotine.
Sazetidine is a partial agonist with much higher affinity than either varenicline or cytisine for (a4b2) 2 b2 and it does not desensitize (a4b2) 2 a4. Therefore, it has a very potent net desensitizing effect on the (a4b2) 2 b2 stoichiometry while allowing nicotine to cause smoldering activation or desensitization of the (a4b2) 2 a4 stoichiometry.
On a3b4 AChRs, nicotine is expected to produce little activation or desensitization at concentrations typically sustained in smokers. Varenicline at submicromolar concentrations will also have limited effect. Cytisine should cause significant desensitization, but little agonist activity, at submicromolar concentrations. Sazetidine at submicromolar concentrations will cause significant smoldering activation as well as desensitization, thereby differing significantly from varenicline and cytisine.
On a7 AChRs, nicotine at concentrations sustained in smokers should have little agonist or desensitizing effect. Varenicline should Figure 7. Short Term Desensitization of a4b2, a3b4 and a7 AChRs by Various Agonists. Responses of a4b2, a3b4 and a7 AChRs to the acute application of ACh (100 mM), nicotine (16 mM), varenicline (4 mM), cytisine (16 mM) and sazetidine-A (62.5 nM) were measured using the FlexStation as described. These drug concentrations were selected because they gave maximum sustained responses to these agonists. Results were expressed as a percentage of maximum response to ACh. Responses were monitored for 10 minutes, and then specific antagonists were added and responses recorded for another two minutes. The antagonists were dihydroberythroidine (DHbE) (1 mM) for a4b2, mecamylamine (MCA (10 mM) for a3b4 and methyllycaconitine (MLA) (10 mM) for a7. These concentrations of antagonists were selected because they were sufficient to inhibit responses to the tested agonists without causing activation themselves. doi:10.1371/journal.pone.0079653.g007 cause significant smoldering activation and desensitizing effects at the concentrations used for smoking cessation therapy. This might contribute to the off target effects which have given it a black box warning of psychopathological effects in some smokers. Cytisine should have little effect on a7 at therapeutic doses. However, sazetidine could have very substantial smoldering agonist effects on a7 at concentrations that would be therapeutically significant. This could produce significant off target effects.
In summary, we have defined the range of concentrations of nicotinic agonists and partial agonists which can sustain smolder-ing activation of human a4b2, a3b4 and a7 AChRs. Further studies are needed to determine the role of smoldering activation not only in nicotine addiction but also in the therapeutic effects of nicotinic agonists and partial agonists.