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42 Nicotinic Receptors with High and Low Acetylcholine Sensitivity: Pharmacology, Stoichiometry, and Sensitivity to Long-Term Exposure to Nicotine

School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, United Kingdom (M.M., I.B.); Eli Lilly and Company Ltd., Lilly Research Centre, Windlesham, United Kingdom (R.Z., E.S.); and Department of Chemistry, University of Chile, Santiago, Chile (B.K.C.)

Received January 28, 2006; accepted May 22, 2006

	Abstract

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4 and 2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the 4to 2 ratio. In this study, injection of oocytes with 1:10 4/2 subunit cDNA ratios favored expression of HS 42 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS 42 receptors. The stoichiometry was inferred from the shifts in the ACh EC₅₀ values caused by Leu to Thr mutations at position 9' of the second transmembrane domain of 4 and 2. The 1:10 injection ratio produced the (4)₂(2)₃ stoichiometry, whereas 10:1 injections produced the (4)₃(2)₂ stoichiometry. The agonists epibatidine, 3-[2(S)-azetidinylmethoxy]pyridine (A-85380), 5-ethoxy-metanicotine (TC-2559), cytisine, and 3-Br-cytisine and the antagonists dihydro--erythroidine and d-tubocurarine were more potent at HS receptors. TC-2559 was more efficacious than ACh at HS receptors but was a partial agonist at LS receptors. Epibatidine was more efficacious than ACh at LS receptors and a partial agonist at HS receptors. Cytisine and 5-halogenated cytisines had moderate efficacy at LS receptors but had almost no efficacy at HS receptors. By exploiting the differential effects of ACh, TC-2559 and 5-I-cytisine we evaluated the effects of long-term exposure to nicotine on HS and LS receptors expressed in Xenopus laevis oocytes after cDNA injections or microtransplantation of 42 receptors assembled in human embryonic kidney 293 cells. We conclude that nicotine up-regulates HS 42 receptors, probably by influencing the assembly of receptors rather than by altering the functional state of LS 42 nAChRs.

The stoichiometry of human 42 nAChRs in neurons has not yet been elucidated. Biochemical (Anand et al., 1991) and electrophysiological (Cooper et al., 1991) approaches have shown that chick 42 nAChRs assemble in a stoichiometry of (4)₂(2)₃ when expressed heterologously in Xenopus laevis oocytes, and it has been proposed that this stoichiometry predominates in the chick brain (Anand et al., 1991; Cooper et al., 1991). Several lines of evidence suggest that 42 nAChR exists in two different stoichiometries in the mammalian brain. First, functional studies have shown biphasic agonist concentration-response curves for stimulation of putative 42 nAChR function in mouse thalamic preparations (Marks et al., 1999; Butt et al., 2002). Such function is not present in the thalamus of 4-/- or 2-/- knockout mice, which also lack high-affinity epibatidine sites (Picciotto et al., 2001). Second, 4 and 2 nAChR subunits expressed heterologously in mammalian cell lines (Buisson and Bertrand, 2001; Nelson et al., 2003; Kuryatov et al., 2005; Vallejo et al., 2005) or X. laevis oocytes (Zwart and Vijverberg, 1998; Zwart et al., 2006) assemble into a mixture of receptors with high and low agonist sensitivity. Functional studies in combination with quantification of methionine-labeled subunits have shown that in stably transfected HEK293 cells expressing human 42 nAChR, the 42 receptor with high agonist sensitivity has a stoichiometry of (4)₂(2)₃ whereas the lower sensitivity receptor has a stoichiometry of (4)₃(2)₂ (Nelson et al., 2003). In these cells, the (4)₃(2)₂ stoichiometry predominates, but long-term exposure to nicotine (Buisson and Bertrand, 2001; Nelson et al., 2003; Kuryatov et al., 2005; Sallette et al., 2005; Vallejo et al., 2005), or boosting the number of 2 subunits by additional transfection or culturing cells at 29°C (Nelson et al., 2003), promotes expression of the high ACh sensitivity (4)₂(2)₃ receptor.

Variation of the 4/2 ratio injected into oocytes produces a multiplicity of 42 nAChR types that cannot be accounted for by the assembly of 4 and 2 subunits into only the (4)₂(2)₃ and (4)₃(2)₂ stoichiometries (Zwart and Vijverberg, 1998; López-Hernández et al., 2004). Such subtype diversity could explain discrepancies in reports of the pharmacological profile of heterologously expressed 42 nAChRs; for example, nicotine has been reported to be a full (Rush at el., 2002) and a partial (Chávez-Noriega et al., 1997) agonist at human 42 nAChR expressed heterogously in X. laevis oocytes. It is clear that if properties of heterologously expressed receptors are to be used to facilitate the identification of native 42 nAChR subtypes or in drug discovery, it is crucially important to use experimental designs that favor the expression of homogeneous populations of 42 nAChR types.

The objective of this work was to heterologously express, in X. laevis oocytes, homogeneous populations of human 42 nAChRs to permit pharmacological comparison in native mammalian neurons and to examine the functional effects of long-term exposure to nicotine on specific functional types of the 42 nAChR. In this study, we showed that human 42 nAChRs expressed in X. laevis oocytes injected with either 1:10 or 10:1 4/2 ratios of cDNAs displayed high or low sensitivity to ACh, respectively. We inserted a hydrophobic to hydrophilic reporter mutation in the middle of the second transmembrane domain (TM2; L9'T as numbered by Miller, 1989) of both subunits to determine the number of each subunit present in the high and low sensitivity 42 receptors. Hydrophobic-to-hydrophilic mutations at 9' (Labarca et al., 1995; Boorman et al., 2000) or other positions (Plazas et al., 2005) in TM2 have been used previously to determine the stoichiometry of other nAChRs. We concluded that injection of oocytes with 1:10 4/2 cDNA ratios favors expression of (4)₂(2)₃ receptors, whereas 10:1 ratios produce (4)₃(2)₂ receptors. We characterized the pharmacological properties of the 42 nAChRs with high and low ACh sensitivity and found remarkable differences between them. By exploiting the different pharmacological profile of the receptors, we evaluated the effects of long-term nicotine exposure on the function and receptor numbers of the 42 nAChRs with high and low sensitivity to ACh.

	Materials and Methods

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Materials. X. laevis frogs were purchased from Horst Kaehler, (Hamburg, Germany). Acetylcholine (ACh), dihydro--erythroidine (DHE), (-)-nicotine, d-tubocurarine, A-85380, and cytisine were purchased from Sigma-Aldrich (St. Louis, MO). TC-2559 was a gift from Targacept Inc. (Winston-Salem, NC). [³H]Cytisine (30.4 Ci/mmol) was from PerkinElmer Life and Analytical Sciences (Boston, MA). For the synthesis of halogenated derivatives, cytisine was purified from the seeds of the Mexican plant Sophora secundiflora using standard methods. Cytisine was halogenated with bromine, iodine monochloride, or chlorine in acetic acid to produce mixtures of 3- and 5-Br-cytisine, 3- and 5-I-cytisine and 3- and 5-Cl-cytisine, respectively. The products of these reactions were separated by column chromatography on silica gel, crystallized to homogeneity, and characterized by ¹H and ¹³C NMR and high-resolution electronimpact mass spectrometry. Definitive structure assignments were based on ¹H-¹H correlation spectroscopy experiments.

Expression Vectors and Site-Directed Mutagenesis. The human 4 and 2 nAChR cDNAs were provided by Professor Jon Lindstrom (University of Pennsylvania, Philadelphia, PA) and subcloned into the pcDNA 3.1(hygro-) expression vector (Invitrogen, Ltd., Paisley, UK) for nuclear injection into oocytes. To minimize differences in the transcription of the two cDNAs that may be brought about by untranslated regions flanking the coding regions of the cDNAs (Briggs et al., 2006), subcloning was performed by polymerase chain reaction using primers annealing at the start and end of the translated sequence of the cDNAs. Using these primers, XbaI and NotI restriction sites were introduced at the 5' and 3' ends, respectively, of both subunit cDNAs for subsequent cloning into the linker region of the expression vector.

A hydrophobic to hydrophilic mutation at position 9' in TM2, 4L283T or 2L274T (Fig. 2A), was introduced in both subunits using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands). The mutant subunits are referred to as 4^LT and 2^LT hereafter. The full-length sequence of both subcloned wild subunit and mutant subunit cDNAs was verified by DNA sequencing (Department of Biochemistry, University of Oxford, Oxford, UK).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Effects of the L9'T mutation on the concentration-response curves of ACh at high (HS) and low (LS) ACh sensitivity 42 nAChRs. A, alignment of the sequences of the second transmembrane domain (TM2) of human 4 and 2 nAChR subunits. Leu at position 9' of TM2 was mutated to Thr in both subunits. B and D, pooled normalized ACh concentration-response data were fitted with the sigmoidal equation under the constraint of equal slopes (nH = 1) to estimate the horizontal distance between the curves as a potency ratio (estimated parameters are shown in Table 1). The data were best fitted to a single sigmoidal equation (p < 0.001-0.0001, F test, four to six cells). C and E, currents through channels spontaneously open (Ispt), determined using 100 µM mecamylamine, and the maximum ACh-activated currents (IACh) were measured and plotted as a ratio. The recordings were carried out at the holding potential of -60 mV. Note that Ispt increased as a function of the number of mutant subunits in the receptors.

Oocytes were impaled by two agarose-cushioned microelectrodes filled with 3 M KCl (0.5-2.0 M) and voltage-clamped at -60 mV using a GeneClamp 500B amplifier and pCLAMP 6 software (Molecular Devices, Sunnyvale, CA). In general, traces were filtered at 1 kHz during recording and digitized at 0.5 to 5 kHz using the Digi-Data 1200 interface (Molecular Devices). All experiments were carried out at room temperature. Agonist concentration-response curves were obtained by normalizing agonist-induced responses to the control responses induced by 1 mM ACh (a near-maximal effective concentration at receptors obtained with 10:1 or 1:1 4/2 cDNA transfecting ratios but an EC₁₀₀ concentration at receptors expressed by oocytes injected with 1:10 4/2 cDNA ratios). A minimum interval of 4 min was allowed between agonist applications because this was found to be sufficient to ensure reproducible recordings. The sensitivity of the receptors to inhibition by the nAChR antagonists DHE and d-tubocurarine was tested by first superfusing the antagonist for 2 min and then coapplying it with an EC₅₀ of ACh (high sensitivity 42 nAChR EC₅₀, 4 µM; low sensitivity 42 nAChR EC₅₀, 88 µM). Antagonist concentration response data were normalized to the appropriate ACh EC₅₀.

Long-Term Nicotine Exposure. The effects of long-term nicotine exposure on the functional expression of highand low-sensitivity 42 nAChRs were examined by determining the effects of ACh, TC-2559, and 5-I-cytisine in oocytes injected with 1:1 4/2 cDNA ratios, unless otherwise mentioned. The reporter mutation approach was not used to monitor changes in the function of 42 nAChRs because the leftward shifts in the ACh concentration-response curves caused by the reporter mutation or by changes in subunit composition would overlap, making it difficult to separate both effects (compare curves in Fig. 2, B and D). Injected oocytes were incubated in 5 µM nicotine-containing Barth's solution for 72 h (or 96 h for some [³H]cytisine binding assays) at 18°C. Exposure to nicotine commenced immediately after injection or 36 h after injection. In agreement with previously published studies (Jia et al., 2003; López-Hernández et al., 2004) the ACh responses of oocytes expressing 42 nAChRs were depressed by long-term exposure to 5 µM nicotine. Therefore, before recordings, oocytes were maintained for 4 h in 15 ml of nicotine-free Barth's solution in a Petri dish (one oocyte per Petri dish) placed on a rotating platform. The nicotine-free medium was replaced with fresh medium every 60 min. This washing procedure permitted sufficient recovery from receptor desensitization to enable reproducible and reliable electrophysiological recordings. The ACh responses of control oocytes (cells not exposed to nicotine) were monitored over the same period as those of nicotinetreated oocytes.

Microtransplantation of Membranes Containing Human 42 nAChR to Oocytes. Membranes of HEK293 cells stably expressing human 42 nAChR (HEK-h42) were injected into the cytoplasm of oocytes using methods reported previously (Miledi et al., 2002; Palma et al., 2003). HEK-h42 cells were grown as described previously (Zwart et al., 2006). Cells (2.4 x 10⁸ cells) were homogenized with 2 ml of a solution containing 200 mM glycine, 150 mM NaCl, 50 mM EGTA, 50 mM EDTA, and 300 mM sucrose, supplemented with protease inhibitors, pH 9 (Palma et al., 2003). The homogenate was first centrifuged for 15 min at 9500g with a TL-100-4 rotor at 4°C in a TL100 centrifuge (Beckman Coulter, Fullerton, CA). The resulting supernatant was centrifuged for 2 h at 100,000g with the same rotor and centrifuge used in the first centrifugation step. The pellet was resuspended in 5 mM glycine to a concentration of 9 to 14 mg of protein/ml. Oocytes were injected with 100 nl of this preparation. Total protein was assayed using the BCA protein assay kit (EMD Biosciences, Inc., Darmstadt, Germany). Injected oocytes were maintained in Barth's medium, and recordings were made 72 h after injection.

[³H]Cytisine Binding Assays. Batches of 20 oocytes expressing 42 receptors were homogenized using a glass homogenizer in 10 ml of a buffer containing 340 mM sucrose, 1 mM EGTA, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride, a serine protease inhibitor. Homogenates were centrifuged at 4°C at 1000g for 10 min. The supernatant was removed for further centrifugation, avoiding disturbance of both the surface lipid layer and the pellet that contained pigment granules, both of which increased nonspecific binding. The supernatant was centrifuged for 1 h at 100,000g. The resulting pellet was resuspended in binding saline (20 µl per oocyte) of the following composition: 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM HEPES, 1% bovine serum albumin (w/v), pH 7.5, plus 1 mM EGTA, 1 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride, supplemented with 10 µg/ml each aprotinin, leupeptin, and pepstatin A to protect the receptors. [³H]Cytisine binding assays were performed by adding a maximal concentration of [³H]cytisine (5 nM) to the resuspended membranes for 75 min at 4°C, and 10 µM nicotine was used to define nonspecific binding. Bound and free fractions were separated by rapid vacuum filtration through GF/B filters (Whatman, Maidstone, UK) presoaked in binding saline with 0.1% polyethylenimine. Radioactivity was determined by liquid scintillation counting using Ecoscint H (National Diagnostics, Atlanta, GA).

Data Analyses. The concentration of ACh that evokes 50% of maximum response [EC₅₀ and 95% confidence interval (CI)] and Hill coefficient (n_H, mean ± S.E.M.) were determined by nonlinear regression. Concentration-response curves were obtained by normalizing the current responses to varying concentrations of ACh to the maximal response observed to a saturating concentration of ACh (1 mM) in each oocyte. Fits to full concentration-response curves for individual oocytes were made independently using Prism 4 (GraphPad Software, San Diego, CA) and then averaged to compare significant differences between groups. Concentration-response data for wild or mutant 42 receptors were fitted using one- and two-component sigmoidal doseresponse equations, Y = I/I_{max Bottom} + (I/I_{max Top} - I/I_{max Bottom})/(1 + 10^{(LogEC50 - X)} x nH) and Y = I/I_{max Bottom} + [(I/I_{max Top} - I/I_{max Bottom}) x Fraction/(1 + 10^{(LogEC50_1 - X) x nH_1})] + [I/I_{max Top} - I/I_{max Bottom}) x (1-Fraction)/(1 + 10^{(LogEC50_2 - X) x nH_2})], respectively, where X is the logarithm of the agonist concentration, Y is the peak amplitude of the normalized current response, I_max is the maximal current, and n_H is the Hill slope. An F test determined whether the one- or two-site model best fit the data; the simpler one-component model was preferred unless the extra sum-of-squares F test had a p value less than 0.05.

To infer the stoichiometry of the high and low ACh sensitivity 42 receptor subtypes, ACh concentration-response data obtained from mutant receptors were analyzed as described previously by Boorman et al. (2000). In brief, ACh concentration-response data were normalized as described previously and then fitted with one- or two-component sigmoidal equations. Fits were carried out constraining the Hill coefficient to 1 to obtain parallel fit curves, as shown in Fig. 2, B and C. The constrained fits were used to estimate the potency ratio (EC₅₀ wild-type receptor/EC₅₀ mutant receptor).

The introduction of a 9' hydrophilic mutation into receptors of the Cys-loop family increases the rate of spontaneous openings (Revah et al., 1991; Chang and Weiss, 1999), which increased with the introduction of progressively higher numbers of mutant subunits (Fig. 2, B and D). The current through the spontaneously open channels (I_spt) in oocytes expressing the mutant 42 receptors was determined (at a holding potential of -60 mV) using 100 µM mecamylamine, a nAChR channel blocker. Spontaneous openings in oocytes expressing wild-type 42 nAChRs were not detected under our experimental conditions.

Binding and concentration-response data were pooled from at least two different batches of oocytes (EC₅₀ and 95% CI). Tests of significance were performed using Student's t test, and p values less than 0.05 were considered significant.

	Results

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Concentration-response curves for whole-cell peak-current responses to ACh were obtained from oocytes injected with 1:1, 1:10, or 10:1 4/2 cDNA ratios (Fig. 1, A-E). Normalized ACh concentration-response data from oocytes injected with 1:1 4/2 cDNA ratios were best fitted to a two-component sigmoidal equation (p = 0.026, F test, n = 10), indicating the presence of two types of 42 nAChRs that differed in their sensitivity to activation by ACh (Fig. 1A). The estimated EC₅₀ (and 95% CI (and n_H ± S.E.M. values for the two components were: high ACh sensitivity component, 1.0 (0.3-2.8) µM and 1.2 ± 0.1; low ACh sensitivity component, 97 (66-142) µM and 1.1 ± 0.3. The relative proportions of the two components were 0.25 ± 0.07 and 0.75 ± 0.09, respectively. These data are comparable with previously reported data concerning ACh effects on oocytes (Zwart and Vijverberg, 1998; Zwart et al., 2006) or mammalian cells (Buisson and Bertrand, 2001; Nelson et al., 2003) coexpressing heterologously high and low ACh sensitivity 42 nAChRs. ACh concentration-response data from oocytes injected with 1:10 or 10:1 4/2 cDNA ratios were best fitted to a one-component sigmoidal equation (p < 0.0001, F test, n = 10), and the slope of the resulting curves was near unity (Fig. 1C; data summarized in Table 3). 42 nAChRs expressed by oocytes injected with the 1:10 4/2 cDNA ratio displayed high sensitivity to activation by ACh (EC₅₀ 4.5 µM), whereas 42 nAChRs from oocytes injected with the 10:1 ratio had lower sensitivity to ACh (EC₅₀ 88 µM). Hereafter, the 42 nAChRs obtained by injecting oocytes with 1:10 or 10:1 4/2 cDNA ratios will be referred to as HS 42 nAChRs or LS 42 nAChRs, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Effects of 4/2 cDNA ratios on the sensitivity of human 42 nACh to activation by ACh. Concentration-response curves (left) for ACh-induced whole-cell current responses (normalized response relative to that at 1 mM ACh) in oocytes injected with a 1:1 ratio of 4/2 cDNAs (, data best fitted to a two-component sigmoidal equation, p = 0.026, F test, n = 10) (A) or 1:10 () or 10:1 () ratios of 4/2 cDNAs (data best fitted to a one-component sigmoidal equation, p < 0.0001, F test, n = 10) (C). Parameters from the fits to the data are presented in Table 3. Also shown (right) are representative traces of whole-cell responses under the indicated conditions of 4 and 2 cDNA injection ratios (1:1, B; 1:10, D; and 10:1, E) and ACh concentration. Note current amplitude and time calibration bars.

View this table: [in this window] [in a new window]   TABLE 3 Functional effects of nicotinic compounds on high (HS) and low (LS) ACh sensitivity 42 nAChRs Concentration-response data were obtained and fitted to one- or two-component sigmoidal equations as described under Materials and Methods. Data were obtained from at least five oocytes from two different donors. Imax and nH values are presented as mean ± S.E.M.

The amplitude of the current responses elicited by maximal concentrations of ACh and maximal [³H]cytisine binding were significantly higher at 42 nAChRs obtained by injecting oocytes with equal amounts of 4 and 2 cDNAs (5.0 ± 0.6 µA and 67 ± 6.4 fmol/oocyte, respectively) than at HS 42 nAChRs (0.4 ± 0.1 µA, p < 0.0001, Student's t test n = 7; 19 ± 3.2 fmol/oocyte, p = 0.026, Student's t test, n = 3) or LS 42 nAChRs (1.5 ± 0.2 µA, p < 0.0001, Student's t test, n = 7; and 29 ± 1 fmol/oocyte, p < 0.0042, Student's t test, n = 3) (Fig. 1, B, D, and E), even though the total amount of cDNA injected using the 1:1; 1:10, or 10:1 ratios was kept constant. One explanation for this finding is that equal or similar levels of 4 and 2 subunits may produce higher levels of a critical heteromeric precursor of 42 nAChRs (e.g., the tetramer 4242; Kuryatov et al., 2005; Sallette et al., 2005) than extreme subunit ratios such as the 1:10 or 10:1 ratios used in this study, resulting in higher levels of mature receptors. It is noteworthy that the amplitude of current responses evoked by 1 mM ACh (Fig. 1, D and E) (p = 0.0004, Student's t test, n = 7) and maximal [³H]cytisine binding (Fig. 6) (p = 0.0046, Student's t test, n = 3) were significantly smaller in HS 42 nAChRs than in LS 42 nAChRs. These results, together with the observation that LS 42 nAChRs predominate under conditions of equal subunit availability, suggest that LS 42 nAChRs may be more efficiently assembled and/or transported to the cell surface than HS 42 receptors. In addition, LS 42 receptors may have a larger unitary current compared with HS 42 receptors. It has been reported previously that high and low agonist sensitivity 42 nAChRs display different unitary conductances (Buisson and Bertrand, 2001; Nelson et al., 2003), although these studies had opposite views on which receptor type has the larger unitary conductance.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Effects of long-term exposure to nicotine on maximal binding of [3H]cytisine to membrane homogenates prepared from X. laevis oocytes heterologously expressing 42 nAChRs. Bar graphs show the effects of 72 h (A) or 96 h (B) exposure to nicotine on maximal binding of [3H]cytisine to oocytes injected with 1:1, 1:10, or 10:1 human 4/2 cDNA ratios. [3H]cytisine binding assays were carried out as described under Materials and Methods. *, p < 0.05; **, p < 0.001, Student's t tests.

View this table: [in this window] [in a new window]   TABLE 1 Effects of TM2 L9'T mutation on the functional effects of ACh on LS 42 nAChR EC50 are the means of EC50 estimates obtained by fitting separately each concentration-response curve with a one-component sigmoidal equation, which gave the best fit for all data sets. Potency ratios were calculated from fits in which curves were constrained to slope equal to 1 and are expressed in relation to receptors containing 5 L9' (which therefore have dose ratio values of 1). Values represent data from three to six experiments (n).

View this table: [in this window] [in a new window]   TABLE 2 Effects of TM2 L9'T mutation on the functional effects of ACh on HS 4 2 nAChRs Oocytes were injected with a 1:10 4/2 cDNA ratio to produce HS 42 nAChRs. EC50 values are the means of EC50 estimates obtained by fitting separately each concentration-response curve with a one-component sigmoidal equation, which gave the best fit for all data sets. Potency ratios were calculated from fits in which curves were constrained to slope equal to 1 and are expressed in relation to receptors containing 5 L9' (which therefore have dose ratio values of 1). Results are from four or five independent experiments (n).

For LS 42 nAChRs, when both subunits carried the mutation, the ACh sensitivity increased 16,800-fold. When 4 carried the mutation, the ACh sensitivity increased 456-fold, whereas when 2 carried the mutation, the increase was only 60-fold. These results show that at the 9' position, both subunits contribute in a multiplicative and symmetric manner to the shifts in ACh EC₅₀ values; therefore, the LS 42 nAChRs contain more 4 than 2 subunits. As reasoned by Boorman et al. (2000) to infer the stoichiometry of 3-containing nAChRs, if the stoichiometry of the LS 42 nAChR is (4)₃(2)₂, then the ³ of the potency ratio obtained when the mutation is carried only by the 4 subunit (i.e., ³456), should be equal to the ² of the potency ratio obtained when the mutation is carried by the 2 subunit (i.e., ² 60). Moreover, the ⁵ of the potency ratio obtained when both subunits carry the L9'T mutation should also be similar to the ³ and ² values. As shown in Table 1, the estimated values for ³, ², and ⁵ are in agreement with this prediction.

For HS 42 nAChRs, there was a 4.2-fold decrease in the value of ACh EC₅₀ when the mutation was carried by the 4 subunit (Table 2) whereas when the 2 subunit carried the mutation there was an 18-fold decrease in the ACh EC₅₀ value. When both subunits carried the L9'T mutation, the ACh EC₅₀ value decreased 137-fold. From these results we concluded that HS 42 receptors contain more 2 than 4 subunits. If the stoichiometry of HS 42 nAChRs is (4)₂(2)₃, then the ² of the potency ratio for HS 4^LT2nAChRs should be approximately the same as the ³ of the potency ratio for HS 42^LT nAChRs, which should also be similar to the ⁵ of the potency ratio for HS 4^LT2^LT nAChRs. Table 2 shows that the estimated values are in accord with the proposed stoichiometry.

In addition to the increase in ACh sensitivity, oocytes expressing mutant LS or HS 42 receptors required a larger holding current to voltage-clamp the membrane at -60 mV compared with oocytes expressing the wild-type receptor. This holding current was blocked by the nAChR ion channel blocker mecamylamine, indicating that it was the result of spontaneously opening mutant 42 nAChRs. Figure 2, C and E, shows the relationship between spontaneous openings and type of mutant subunit present in LS or HS 42 nAChRs, respectively. The degree of spontaneous openings increased as a function of the number of mutant subunits in the heteropentamers, but the effect was most marked when the receptor was of the LS type. Hydrophilic substitution of the conserved M2 leucine also created spontaneous openly GABA-A receptors (Chang and Weiss, 1999). However, the mutation did not produce spontaneously opening 343or 34 nAChRs (Boorman et al., 2000). This suggests a difference in the degree of perturbation produced by the mutation in the activation of the two nAChR types.

To test the validity of the proposed subunit combination for the HS and LS 42 nAChRs, we plotted the logarithm of the ACh EC₅₀ against the presumed number of mutations in each assembled receptor complex. If the observed changes in ACh sensitivity brought about by the L9'T mutation depend only on the number of copies of the mutated subunits in the receptor, the plot should be linear. As shown in Fig. 3, A and C, the relationship between the ACh EC₅₀ of the different combinations of LS or HS 42 nAChRs and the number of putative L9'T mutations in each receptor type was linear. These results, therefore, are in accord with our conclusion that the stoichiometries of 42 nAChRs expressed by X. laevis oocytes after injections with 1:10 or 10:1 4/2 cDNA ratios are (4)₂(2)₃ and (4)₃(2)₂, respectively. Note that the logarithm of fractional spontaneous openings was also linearly related to the number of copies of the mutated subunits in the LS or HS receptors (Fig. 3, B and D)

View larger version (31K): [in this window] [in a new window]   Fig. 3. Relation between the putative number of L9'T mutations in 42 nAChRs and the ACh EC50 and currents through mutant 42 mAChRs spontaneously open. A and C, ACh EC50 of 42 nAChRs and their putative number of L9'T mutations. ACh EC50 values were plotted on a log scale against the number of mutant subunits in either high-sensitivity (HS) or low-sensitivity (LS) 42 nAChRs. Note the approximate linearity of the decline in EC50 for both types of receptor. The graphs suggest that the 4 subunit is represented three times in the LS receptor and twice in the HS receptor, whereas the 2 subunit is represented twice in the LS receptor and three times in the HS receptor. Insets show the subunit arrangement for each 42 nAChR subtype. Arrows indicate the agonist binding sites located at interfaces. B and D, plot of the relationship between the number of mutant subunits in the 42 nAChRs and logIspt/IACh.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Concentration-response curves of nicotinic agonists and antagonists on the function of 42 nAChR types. Nicotinic agonists (A) and antagonists (B) had different effects at 42 nAChRs with high (HS) and low (LS) ACh sensitivity. A, concentration-response curves of nicotinic agonists on the function of HS () and LS () 42 nAChRs. Note that all agonists tested activated HS and LS 42 nAChRs with the exception of 5-halocytisines, which activated current responses at LS 42 nAChRs only. Estimated EC50 and Hill coefficients for agonists are provided in Table 3. B, concentration-response curves of nicotinic antagonists on EC50 ACh responses of HS () and LS () 42 nAChRs. Estimated IC50 for antagonists are provided in text. Agonist or antagonist data were analyzed as described under Materials and Methods using a monophasic or biphasic sigmoidal equation. Data points represent the mean of 5 to 10 experiments carried out using at least two different batches of oocytes.

Inward currents elicited by EC₅₀ of ACh at either HS or LS 42 nAChRs were inhibited by the 42 nAChR antagonist DHE in a concentration-dependent and monophasic manner (p < 0.0001, F test, n = 6) (Fig. 4B). HS 42 receptors were more sensitive to inhibition by DHE [IC₅₀, 17 (14-19) nM] than LS 42 nAChRs [IC₅₀, 410 (361-485) nM]. The current responses of HS or LS 42 nAChR to EC₅₀ concentrations of ACh were also inhibited by d-tubocurarine, a nonspecific nicotinic competitive antagonist. At both receptor types, the effects of d-tubocurarine were concentration-dependent and monophasic (p < 0.0001, F test, n = 5). HS 42 nAChRs were inhibited with an estimated IC₅₀ value of 0.28 (0.22-0.35) µM. Higher concentrations of d-tubocurarine were required to inhibit LS 42 nAChRs [IC₅₀ = 6.5 (3.4-12) µM].

Overall, the findings of these studies give additional support to our view that the 1:10 and 10:1 4/2 subunit cDNA injection ratios produce homogeneous populations of HS and LS 42 nAChRs, respectively. Although, in principle, other subunit combinations may form functional receptors [e.g., 4(2)₄;López-Hernández et al., 2004], in view of the pharmacological differences between the HS and LS 42 nAChRS, it is unlikely that other putative subunit combinations would display ligand sensitivities that were so similar to those of the HS and LS 42 nAChR types as to render them indistinguishable from them.

Nicotine Up-Regulates HS 42 nAChRs in X. laevis Oocytes. When mammalian cell lines heterologously expressing a mixture of high and low ACh sensitivity 42 nAChRs are exposed to nicotine for a long period of time, there is a selective increase in the function of 42 nAChRs with high ACh sensitivity (Buisson and Bertrand, 2001; Nelson et al., 2003; Kuryatov et al., 2005; Sallette et al., 2005; Vallejo et al., 2005). To determine whether human HS 42 receptors in X. laevis oocytes are up-regulated similarly, oocytes injected with the 1:1 4/2 cDNA ratio were exposed to 5 µM nicotine immediately after injection for a period of 72 h and were washed for 4 h; the responses to ACh, TC-2559, and 5-I-cytisine were then determined. When oocytes injected with equal amounts of 4 and 2 cDNAs were exposed to nicotine immediately after injection (i.e., before any intermediate species or mature receptor could have assembled; Kuryatov et al., 2005; Sallette et al., 2005), the expressed 42 nAChRs displayed only one EC₅₀ value for ACh. The ACh EC₅₀ value estimated from the monophasic curve (p < 0.005, F test, n = 5) was 3 (1.83-7.63) µM, which was not significantly different from the ACh EC₅₀ at HS nAChRs expressed by oocytes injected with the 1:10 4/2 cDNA ratio. We next evaluated the effects of TC-2559, which is more efficacious than ACh at HS 42 nAChRs (but of similar potency at both receptors) and the effects of 5-I-cytisine, which activates only LS 42 nAChRs (Table 3). As shown in Fig. 5B, long-term exposure to nicotine increased the maximal response to TC-2559 by 540%. As expected from the negligible difference in the EC₅₀ values of TC-2559 at HS and LS 42 nAChRs (Table 3), concentration-response curves for TC-2559 at control and nicotine-exposed oocytes were monophasic (p < 0.001, F test, n = 5), and the estimated EC₅₀ values [control, 2.4 (1.0-5.4) µM; nicotine-treated, 5.4 (3.4-10) µM] were not significantly different from each other. In contrast, nicotine decreased the relative efficacy of 5-I-cytisine from 50 to 0.94% (Fig. 5C). These results show that in response to nicotine, oocytes favored expression of HS 42 nAChRs, even though they were injected with 4/2 cDNA ratios that produced a mixture of 42 nAChRs enriched in LS receptors in oocytes not exposed to nicotine.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Effects of long-term nicotine exposure on oocytes injected with a 1:1 4/2 subunit cDNA ratio. A, in oocytes injected with 1:1 4/2 subunit cDNA, exposed to nicotine immediately and for 72 h subsequently, then studied electrophysiologically, the concentration-response curve of ACh in oocytes was monophasic (p < 0.005, F test, n = 5) (). In contrast, the effects of ACh in oocytes that were not exposed to nicotine () were biphasic (p < 0.0026, F test, n = 10). B, concentration-response curve for TC-2559 effects at oocytes injected with 1:1 4/2 cDNA ratio () and exposed to nicotine immediately after injection and for 72 h subsequently. Control oocytes () were injected with 1:1 4/2 subunit cDNA ratio but were not exposed to nicotine. C, concentration-response curves for 5-I-cytisine effects at oocytes injected with a 1:1 4/2 cDNA ratio () and exposed to nicotine immediately after injection and for 72 h subsequently. Control oocytes () were injected with the same amount of subunit cDNAs but were not exposed to nicotine. D, exposure to nicotine 36 h after injection with a 1:1 4/2 cDNA ratio did not change the biphasic nature of the ACh concentration-response curve, although it increased the high sensitivity component of the biphasic ACh concentration-response curve from 0.25 to 0.49 of the total receptor population. E, bar graph showing that exposure to nicotine 36 h after injection with a 1:1 4/2 cDNA ratio increased the efficacy of TC-2559 by 44%, whereas the efficacy of 5-I-cytisine remained unchanged.

We next evaluated how oocytes expressing a mixture of 42 nAChRs dominated by the LS receptor type modify, in response to nicotine, the expression of HS and LS 42 receptors. For these experiments, oocytes injected with 1:1 4/2 cDNA ratios were exposed to nicotine 36 h after injection, when HS and LS receptor function was clearly detected as shown by the biphasic ACh concentration-response curves obtained at 36 h after injection (Fig. 5D). Oocytes were exposed to nicotine for 72 h. As shown in Fig. 5D, exposure to nicotine 36 h after injection increased the fraction of nAChRs with higher sensitivity to ACh from 0.25 ± 0.07 to 0.49 ± 0.1 of the total in oocytes. The EC₅₀ values for the HS and LS components were 1 (0.13-7) µM and 80 (21-136) µM, which were comparable with those obtained from control oocytes. These results further support the proposal that in response to nicotine incubation, cells specifically up-regulate 42 nAChRs with high ACh sensitivity (Buisson and Bertrand, 2001; Nelson et al., 2003; Kuryatov et al., 2005; Sallette et al., 2005; Vallejo et al., 2005). We also evaluated the effects of TC-2559 and 5-I-cytisine. As shown in Fig. 5E, TC-2559 efficacy increased significantly by 44% [control oocytes, 0.50 ± 0.06; nicotine-incubated oocytes, 0.72 ± 0.009 (p = 0.026, Student's t test, n = 4)], but the efficacy of 5-I-cytisine at nicotine-incubated oocytes (0.44 ± 0.09; n = 4) was not significantly different from that of control oocytes (0.42 ± 0.04; n = 3) (Fig. 5E). Thus, our results suggest that nicotine up-regulates expression of HS 42 nAChRs without affecting the expression of LS 42 nAChRs.

We performed additional experiments to assess whether nicotine affects the functional expression of 42 nAChRs obtained by injecting oocytes with 1:10 or 10:1 4/2 cDNA ratios. We were surprised to find that, in contrast to the 42 nAChRs expressed by oocytes injected with 1:1 4/2 cDNA ratios, exposure to nicotine did not lead to a significant change in the relative efficacy of 5-I-cytisine or TC-2559 at LS or HS 42 nAChRs produced using injections with 10:1 or 1:10 subunit cDNA ratios (data not shown). One explanation for these results could be that the 1:10 or 10:1 cDNA ratios are too extreme for nicotine to exert measurable changes in the functional expression of HS or LS 42 nAChRs. Because nicotine-induced nAChR up-regulation is faster in cells with higher levels of receptor expression (Nashmi et al., 2003), up-regulation under extreme subunit ratios, which leads to low receptor expression levels, may be slower than under conditions of balanced subunit abundance. In this case, the effects of nicotine should become detectable with longer periods of incubation. To test this hypothesis, we evaluated binding of 5 nM [³H]cytisine to membrane homogenates prepared from oocytes that were incubated in nicotine for 72 h or 96 h after injection with 1:1, 1:10, or 10:1 subunit cDNA ratios. As shown in Fig. 6A, nicotine increased binding of 5 n M[³H]cytisine to membranes from oocytes injected with 1:10 or 10:1 4/2 cDNA ratios by less than 1%. In contrast, [³H]cytisine binding sites in membrane homogenates from oocytes injected with equal amounts of 4 and 2 subunit cDNAs were up-regulated from 65 ± 5 fmol/oocyte to 85 ± 3 fmol/oocyte. Incubation in nicotine for 96 h further increased [³H]cytisine binding to oocytes injected with 1:1 subunit cDNA ratios to 125 ± 7 fmol/oocyte (Fig. 6B). Maximal [³H]cytisine binding to membranes from oocytes injected with 1:10 cDNA ratios was also increased after 96-h incubation in nicotine (control oocytes, 19 ± 4 fmol/oocyte; nicotine-incubated oocytes, 28 ± 3 fmol/oocyte) (Fig. 6B). Thus, longer periods of incubation were required to up-regulate HS 42 nAChRs expressed under conditions of subunit imbalance. It is noteworthy that maximal [³H]cytisine binding to membrane homogenates prepared from oocytes injected with 10:1 4/2 cDNA ratios was not affected by 96-h nicotine incubation (29 ± 5 fmol/oocyte compared with 32 ± 7 fmol/oocyte in control oocytes) (Fig. 6B).

The preceding results generally support the view that nicotine selectively up-regulates HS 42 nAChRs (Buisson and Bertrand, 2001; Nashmi et al., 2003; Nelson et al., 2003; Kuryatov et al., 2005; Sallette et al., 2005; Vallejo et al., 2005). It has been suggested that nicotine up-regulates nAChRs by enhancing receptor assembly (Wang et al., 1998; Nashmi et al., 2003; Nelson et al., 2003; Kuryatov et al., 2005; Sallette et al., 2005). An alternative view is that longterm exposure to nicotine alters the functional state of 42 nAChR present in the plasma membrane (Buisson and Bertrand, 2001; Vallejo et al., 2005). A straightforward approach to further test these views in X. laevis oocytes would be to examine the effects of long-term exposure to nicotine on 42 nAChR assembled in HEK-h42 cells and then microtransplanted into X. laevis oocytes (Palma et al., 2003). Microtransplanted human 42 nAChRs have been shown to display identical pharmacological and functional properties as their in situ counterparts (Palma et al., 2003), which supports their use as a model of mature 42 nAChRs. If nicotine up-regulates 42 nAChRs by increasing assembly of receptors, then the functional state of microtransplanted 42 nAChRs should not be affected by long-term exposure to nicotine. But, if nicotine alters the functional state of 42 nAChRs, then microtransplanted receptors exposed to nicotine in oocytes should have altered sensitivity to functional activation. Figure 7A shows that human 42 nAChRs isolated from HEK-h42 cells and then microtransplanted into oocytes were of low and high ACh sensitivity types. Concentration-response curves for ACh were best fit to the two-component sigmoidal equation (p < 0.001, F test, n = 6). Calculations yielded the following EC₅₀ values. 42 nAChRs with high ACh sensitivity EC₅₀ = 1.6 (0.5-7.4) µM. 42 nAChRs with low ACh sensitivity EC₅₀ = 199 (158-253) µM. The lower sensitivity component comprised 78% of the overall receptor population. The microtransplanted receptors, therefore, had properties similar to those of the human 42 nAChRs expressed by our HEK293 cell line (Zuo et al., 2003) and other HEK293 cell lines (Buisson and Bertrand, 2001; Nelson et al., 2003; Kuryatov et al., 2005) stably expressing human 42 nAChRs. We then examined the sensitivity to activation of the microtransplanted receptors after 72 h exposure to nicotine. Figure 7B shows that the concentration-response curve for ACh at microtransplanted 42 nAChRs exposed to nicotine was not significantly different from that of microtransplanted receptors that were not exposed to nicotine treatment. The ACh concentration-response curves of receptors exposed to nicotine were best fitted to a two-component sigmoidal equation (p < 0.001, F test, n = 6). The estimated EC₅₀ values were: high ACh sensitivity EC₅₀ = 1 (0.3-1.2) µM; low ACh sensitivity EC₅₀ = 197 (154-251) µM. Furthermore, the relative efficacies of TC-2559 or 5-I-cytisine were not changed by nicotine (Fig. 7C). These results are in accord with the first prediction.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Effects of long-term nicotine exposure on oocytes expressing microtransplanted human 42 nAChRs. The receptors were assembled and expressed first in HEK293 cells stably expressing human 42 nAChRs. Oocytes expressing microtransplanted 42 nAChRs were incubated in nicotine for 72 h and then used for electrophysiological experiments as described under Materials and Methods. A, ACh concentration response data for both control and nicotine-treated oocytes were best fitted to a two-component sigmoidal equation (p < 0.001, F test, n = 6). Control oocytes, ----; nicotine-incubated oocytes: ——. Inset shows a typical current response to 1 mM ACh in HEK293 cell stably expressing human 4