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Roles of Accessory Subunits in 4β2^* Nicotinic Receptors

Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania

Received February 29, 2008; accepted March 31, 2008

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Accessory subunits in heteromeric nicotinic receptors (AChRs) do not take part in forming ACh binding sites. 5 and β3 subunits can function only as accessory subunits. We show that both 5 and β3 efficiently assemble in human 4β2^* AChRs expressed in permanently transfected human embryonic kidney (HEK) cell lines. Only (4β2)₂5, not (4β2)₂β3 AChRs, have been detected in brain. The 4β25 line expressed 40% more AChRs than the parent 4β2 line and was equally sensitive to up-regulation by nicotine. The 4β2β3 line expressed 25-fold more AChRs than the parental line and could not be further up-regulated by nicotine. Relative sensitivity to activation by ACh depends on the accessory subunit, β2 conferring the greatest sensitivity, 5 less, and β3 and 4 much less. Accessory subunits form binding sites for positive allosteric modulators, as illustrated by the observation that 5 conferred high sensitivity to galanthamine. In the presence of 5 or β3, stable, partially degraded, dead end intermediates accumulated within the cells. These may have the form 54β25. The efficiency with which 5 and β3 assemble with 4 and β2 and the necessity of avoiding formation of potentially toxic intermediates may explain why 5 and β3 seem to be transcribed at low levels in brain. Autosomal dominant nocturnal frontal lobe epilepsy can be caused by the 4 mutation S247F. This mutant did not produce functional AChRs unless cells were cotransfected with 5, β3, or 6 to replace 4 as accessory subunit.

When expressed in permanently transfected human cell lines, most human 4β2 AChRs are in the (4β2)₂4 stoichiometry, which has low sensitivity to ACh and rapid desensitization relative to the (4β2)₂β2 stoichiometry (Nelson et al., 2003). Nicotine binds to partially assembled AChRs, acting as a pharmacological chaperone to selectively increase assembly of the (4β2)₂β2 stoichiometry (Kuryatov et al., 2005; Sallette et al., 2005). This stoichiometry has high sensitivity to ACh and slow desensitization.

4β2^* AChRs are the major brain subtypes with high affinity for nicotine, and 11 to 37% of these, depending on brain region, are (4β2)₂5 AChRs (Gerzanich et al., 1998; Brown et al., 2007; Gotti et al., 2007; Mao et al., 2008). Knockout of 5 AChRs in mice reduced activation of high-sensitivity brain AChRs without reducing the total number of AChRs (Brown et al., 2007) and caused resistance to nicotine-induced seizures and hypolocomotion (Salas et al., 2003; Kedmi et al., 2004).

Human (4β2)₂5 AChRs have the high sensitivity to ACh of (4β2)₂β2 AChRs, but higher permeability to Ca²⁺ when expressed in Xenopus laevis oocytes using linked 4 and β2 subunits in combination with free 5 subunits to force formation of this stoichiometry (Tapia et al., 2007). In this system, (4β2)₂β3 and (4β2)₂4 AChRs have low sensitivity to ACh but high permeability to Ca²⁺.

5 subunits in human 3^* AChRs expressed in X. laevis oocytes increased the Ca²⁺ permeability and desensitization rates of all 3 AChRs (Gerzanich et al., 1998). 5 increased the sensitivity of 3β2 but not 3β4 AChRs to activation by ACh. When human 3^* AChRs were expressed in permanently transfected HEK cell lines, expression in the 3β25 line was 2.8-fold greater than the 3β2 line. Both 3β2 and 3β25 lines were up-regulated by nicotine, but 3β4 and 3β45 were not (Wang et al., 1998).

β3-containing AChRs are located in aminergic neurons in association with 6 subunits, and have been found as (6β2)₂β3, (6β4)₂β3, and (4β2)(6β2)β3 AChRs (Champtiaux et al., 2003; Gotti et al., 2007; Salminen et al., 2007). Because ventral tegmental area neurons (which are involved in addiction to nicotine) and substantia nigra neurons (which are involved in Parkinson's disease) express 4, β2, β3, and 6 subunits, (4β2)₂β3 AChRs should have the opportunity to be formed but have not been immunoisolated from brain (Gotti et al., 2007; Perry et al., 2007; Mao et al., 2008). Presynaptic (4β2)(6β2)β3 AChRs modulate the release of dopamine and neuroprotection by nicotine, are exceptionally sensitive to activation by nicotine, and are thought to be especially important in Parkinson's disease and its primate models (Quik et al., 2007; Salminen et al., 2007).

β3 subunits expressed in permanently transfected HEK cell lines promote assembly of (6β2)₂β3 and (6β4)₂β3 AChRs with increased sensitivity to up-regulation by nicotine (Tumkosit et al., 2006).

Autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE) can be caused by the 4 mutation S247F in which a small hydrophilic serine in the M2 sequence lining the channel is replaced by a bulky hydrophobic phenylalanine (Klaassen et al., 2006; Teper et al., 2007). When this mutant is expressed in X. laevis oocytes at subunit mRNA ratios resulting primarily in the (4β2)₂β2 stoichiometry, functional AChRs are formed that lack Ca²⁺ permeability, but coexpression with 5 restores Ca²⁺ permeability (Kuryatov et al., 1997). When expressed in a cell line in which the (4β2)₂4 stoichiometry predominates, no function was observed, presumably because the presence of three phenylalanines blocks the channel but the mutant AChRs are expressed efficiently and nicotine increases their assembly, as with wild-type AChRs (Kuryatov et al., 2005).

Here we report the properties of (4β2)₂5 and (4β2)₂β3 AChRs expressed in permanently transfected HEK cell lines, demonstrating effects of accessory subunits on ACh assembly, sensitivity to activation by agonists, and modulation by allosteric modulators. We also report that replacement of the 4 accessory subunit in the ADNLFE cell line with other AChR subunits permits ion channel function and alters sensitivity to activation.

	Materials and Methods

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cDNAs and Chemicals. Human 4 and β2 cDNAs were cloned in this lab as described previously (Kuryatov et al., 1997; Wang et al., 1998). The cDNA for human 5 was provided by Dr. F. Clementi (CNR University of Milan, Milan, Italy) and subcloned in pCEP4 vector (Invitrogen) (Wang et al., 1998). Human β3 was obtained from Christopher Grantham (Janssen Research Foundation, Beerse, Belgium) and subcloned into pCEP4/Hygromycin(+) for transfection using HindIII and XhoI restriction enzymes. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted.

Tissue Culture and Transfection. The HEK tsA201 parental cell line expressing human 4β2 AChRs was described previously (Nelson et al., 2003; Kuryatov et al., 2005). All cell lines were maintained in Dulbecco's modified Eagle's medium (high glucose; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Invitrogen) at 37°C, 5% CO₂ at saturating humidity.

For transient transfection, 100-mm dishes of 25% confluent 4β2 cells were transfected with 6 µg of β3 or 5 cDNAs using the FuGENE 6 DNA transfection kit (Roche Diagnostics, Indianapolis, IN). After 48 h, cells were collected using ice-cold PBS, and AChRs were extracted.

For permanent transfection, 35-mm dishes of 50% confluent 4β2 cells were transfected with β3 or 5 cDNAs using the FuGENE 6 DNA transfection kit (Roche Diagnostics). Hygromycin (Roche Diagnostics) was added at 0.1 mg/ml for 5 and β3 selection, 0.5 mg/ml Zeocin (Invitrogen) was added for 4 selection, and 0.6 mg/ml G418 (Invitrogen) was added for β2 selection. The transfected cells were passed onto 10-cm dishes before they were passed and plated on a 96-well plate for serial dilution. The 96-well plate was checked for the growth of single colonies; after the colony occupied approximately a quarter of the size of the well, it was plated onto a 24-well plate and then passed to three 35-mm dishes. Each promising clone was then tested to determine how much AChR was present. Screening for cells and extraction of stable clones continued as described previously by Tumkosit et al. (2006). Solid-phase assays for β2-containing AChRs were performed with mAb 295-coated wells, and assays for 5 and β3 containing AChRs were performed with mAb 210-coated wells.

Antiserum and mAbs. A rat antiserum to bacterially expressed 4 subunit sequences (excluding the transmembrane domains) was raised as described previously (Kuryatov et al., 2000). The rat mAb 210 binds to the main immunogenic region of human 1, 3, 5 (Lindstrom, 2000), and β3 (Tumkosit et al., 2006). The rat mAb 295 binds to the extracellular domain of native β2 subunits with high affinity only when they are associated with 3, 4, or 6 subunits (Lindstrom, 2000).

AChR extracts were incubated in mAb-coated microtiter wells for solid-phase radioimmunoassay, or with mAb-coupled to activated CH-Sepharose (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) for purifying AChRs for use in immunoblot assays or loaded directly onto 5-ml sucrose gradients [5-20% sucrose (w/w)] for sedimentation analysis (Kuryatov et al., 2005).

For immunoprecipitation of AChRs with subunit-specific antibodies, the extract was incubated overnight with mAb or antiserum in the presence of [³H]epibatidine (2 nM). The AChR-antibody complexes were immunoprecipitated with sheep anti-rat IgG for rat antibodies. [³H] Epibatidine-labeled AChRs in the pellet were quantified using liquid scintillation counting. Nonspecific precipitation was measured using either normal mouse serum or normal rat serum.

AChR Extraction and Determining 5 and β3 Incorporation. Cells from which AChRs were to be extracted were collected in ice-cold PBS (100 mM NaCl and 10 mM sodium phosphate, pH 7.4) then centrifuged at 13,000g for 15 min in Eppendorf tubes with 1 ml of buffer A (50 mM NaPO₄, pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride). The pellets were resuspended in buffer A plus 2% Triton X-100 and incubated for 1 h at room temperature to solubilize AChRs. Insoluble material was removed by centrifugation at 13,000g for 15 min. Total protein concentration of solubilized AChRs was determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL).

The most stable 4β2β3 clones and 4β25 clones were selectively screened and tested for high expression of β3 and 5 based on liquid phase radioimmune assays with mAb 295 and mAb 210 as described previously (Kuryatov et al., 2005; Tumkosit et al., 2006). AChR-antibody complexes were immunoprecipitated with sheep anti-rat IgG. For immunoprecipitation of AChRs with subunit-specific antibodies, the extract was incubated overnight with mAb or antiserum in the presence of [³H]epibatidine (2 nM) (PerkinElmer Life And Analytical Sciences, Waltham, MA). [³H]Epibatidine-labeled AChRs in the pellet were quantified using liquid scintillation counting. Nonspecific precipitation was measured using normal rat serum.

Sucrose Gradients. Aliquots of 150 µl of cell extract in 2% Triton X-100 in buffer A were layered onto 11.3 ml of linear 5 to 20% sucrose gradients (w/v) in 0.5% Triton X-100 solution of PBS, 5 mM EDTA, 5 mM EGTA, and 1 mM NaN₃ at pH 7.5. The gradient was centrifuged for 16 h at 40,000 rpm in a Beckman SW41 rotor. An aliquot (1 µl) of 2 mg/ml purified Torpedo californica electric organ AChR was added to the cell extract as an internal sedimentation standard. After centrifugation, 17-drop fractions were collected from the bottom. Immulon 96-well 4HBX plates (Thermo Fisher Scientific, Walthman, MA) were coated with mAb 295 to detect β2 subunits, mAb 210 to detect 5 or β3 subunits, or mAb 299 or mAb 371 to detect 4 subunits. Aliquots (20 µl) from each gradient fraction were added to appropriate wells to detect epibatidine binding or -bungarotoxin binding.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Immunoisolation shows that nearly all of the AChRs expressed by the 4β25 and 4β2β3 cell lines contain the expected accessory subunit. mAbs 295 and 210 were coupled at 2 mg/ml to activated CH-agarose 4B. Triton X-100 extracts containing 15 and 30 pM AChR for 4β25 and 100 and 200 pM AChR for 4β2β3 cell lines were labeled with 2 nM [3H]epibatidine and assayed by immune precipitation with mAb 295. Aliquots of extract (20 µl) were adsorbed overnight on a shaker at 4°C with aliquots (20 µl) of mAb agarose, then the supernatants were re-assayed the next day.

Binding of [³H] Epibatidine. Surface expression in 4β25 cells was determined similarly to Kuryatov et al. (2005). Surface expression of 4β2β3 cells has to be done on collagen-coated 24-well plates (BD Discovery Labware, Bedford, MA) because of low adhesion of this cell line. When the 4β2β3 cells reached more than 50% confluence, 0.5 nM [³H]epibatidine was added to wells to label AChRs. Binding to AChRs in the cell surface was inhibited by 1 mM butyrylcholine chloride (Sigma-Aldrich), a membrane impermeable quaternary amine, to determine the internal pool of AChRs. Nonspecific binding was determined by addition of 100 µM nicotine. After incubation for 30 min on ice, the cells were washed three times with 0.5 ml of Dulbecco's modified Eagle's medium and dissolved in 200 µl of 0.1 N NaOH. The bound radioactivity was determined in Eppendorf tubes with 1 ml per tube of OptiPhase "Supermix" scintillation fluid using a 1450 Trilux Microbeta liquid scintillation counter (Perkin-Elmer Life and Analytical Sciences)

Up-Regulation of Epibatidine Binding Sites In Stably Transfected Cell Lines. Cells were plated in 100 µl of medium at a density of 70,000 to 100,000 cells per well on 96-well white clear-bottomed plates (Corning Incorporated, Corning, NY). The next day, nicotine was added. After incubation for 24 h, cells were fixed by adding 100 µl of 4% phosphate buffered formaldehyde (Fisher Scientific, Fair Lawn, NJ) per well for 1 h. Then AChRs were measured using [³H]epibatidine as described above.

FLEXstation Experiments. AChR function was determined in the cell lines using a FLEXstation II (Molecular Devices, Sunnyvale, CA) bench-top scanning fluorometer as described by Kuryatov et al. (2005). The day before the experiment the cells were plated at 100,000 cells/well on poly(D-lysine)-coated black-walled/clear-bottomed 96-well plates (BD Biosciences). Membrane potential and Ca²⁺ assay kits (Molecular Devices, Sunnyvale, CA) were used according to the manufacturer's protocol. Serial dilutions of drugs were prepared in V-shaped 96-well plates (Fisher Scientific Co., Pittsburgh, PA) and were added in separate wells at a rate of 20 µl/s during recording. Each point on the curves represents the average of three to four responses from different wells. The Hill equation was fitted to the concentration-response relationship using a nonlinear least-squares error curve-fit method (Kaleidagraph; Synergy Software, Reading, PA): I(x) = I_max [xⁿ/(xⁿ + EC₅₀n)], where I(x) is the current measured at the agonist concentration x, I_max is the maximal concentration for the half-maximal response, and n is the Hill coefficient.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Construction of Human 4β25 and 4β2β3 AChR-Expressing Cell Lines in tsA201 HEK Cells. As expected, transient transfection of tsA201 HEK cells with the 4β2 subunit combination resulted in functional AChRs, whereas 5 and β3 acted as obligate accessory subunits and did not form epibatidine binding sites when expressed in 4β3, 45, 5β2, or β3β2 combinations (data not shown).

Construction of permanent cell lines started with the 4β2 AChR cell-line described previously (Nelson et al., 2003; Kuryatov et al., 2005). This was transfected with Hu5/pCEP4 or Huβ3/pCEP4 to produce lines expressing 5 or β3 accessory subunits. Total AChR expression was measured by immunoisolation of [³H]epibatidine-labeled AChRs using mAb 295 to β2 subunits. Assays of incorporation of 5 and β3 used mAb 210, which was made to the main immunogenic region of 1 subunits through immunization of rats with bovine muscle AChR (Lindstrom, 2000) but also cross-reacts with human 1 (Lindstrom, 2000), 3 (Wang et al., 1998), 5 (Kuryatov et al., 1997), and β3 (Tumkosit et al., 2006). Adsorption of AChR extracts with mAb 210 coupled to Sepharose beads could adsorb virtually all of the AChRs from the cell lines, showing that virtually all of the AChRs incorporated either 5 or β3 subunits (Fig. 1). This also implies that 5 and β3 were expressed in amounts nearly equal to or greater than the amount of 4 and β2.

The 4β25 line expressed substantial amounts of [³H]epibatidine binding sites (1.75 ± 0.25 pmol/mg protein), approximately 40% more than the amount expressed by the parent 4β2 cell line (1.25 ± 0.35 pmol/mg protein). The 4β2β3 line expressed 25-fold more AChR (30 ± 10 pmol/mg protein). Thus, both 5 and β3 are efficiently incorporated into 4β2^* AChRs. β3 seems to substantially promote assembly of AChRs with the 4β2 line, even more than it does in the cases of 6β2 and 6β4 AChR cell lines (2- to 6-fold) (Tumkosit et al., 2006). Transient transfections of the 4β2 cell line with either 5 or β3 (resulting in incorporation levels of 29 and 49%, respectively) did not significantly increase the total amount of AChR. However, in two other permanently transfected cell lines, (4β2)₂β3 AChRs were expressed in similarly remarkably high levels. One of these lines was the 6β2β3 line described in Tumkosit et al. (2006) transfected with 4, and the other was an 46β2 line transfected with β3. Thus, although there may be selection biases associated with cloning a particular line, after long-term selection, the presence of both 4 and β3 (expressed in excess) was associated with very high levels of (4β2)₂β3 AChR expression but not of greatly increased amounts of 6^* AChRs when 6 was also present.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Activation of AChRs in the 4β25 and 4β2β3 cell lines assayed using a Ca2+-sensitive fluorescent indicator in a FLEXstation. A-C, dose/response curves for 4β25 cells. D-F, dose/response curves for 4β2β3 cells.

As expected for a cell line expressing on its surface exclusively (4β2)₂5 AChRs, there were single component dose/response curves for activation by agonists (Fig. 2, A-C). (4β2)₂5 AChRs in the cell line were 46-fold more sensitive to ACh than were (4β2)₂4 AChRs but 5-fold less sensitive than were (4β2)₂β2 AChRs (Tables 1 and 3; Fig. 2. A-C). Sensitivity to activation by nicotine of (4β2)₂5 AChRs was 3-fold less than the (4β2)₂β2 stoichiometry but 7-fold more than the (4β2)₂4 AChRs. Cytisine behaved as a 25% agonist on (4β2)₂5 AChRs in the cell line. Cytisine has been reported to not act as an agonist on the (4β2)₂β2 stoichiometry and to act as a 22% partial agonist on the (4β2)₂4 stoichiometry expressed in X. laevis oocytes using high ratios of either β2 or 4 to produce one stoichiometry or the other (Moroni et al., 2006). In our hands, using linked 4 and β2 subunits plus free β2, 5, or 4 subunits to produce defined AChR stoichiometries expressed in X. laevis oocytes, cytisine shows 3.6% efficacy on (4β2)₂β2 AChRs, 8.3% efficacy on (4β2)₂5 AChRs, and 22% efficacy on (4β2)₂4 AChRs (data not shown). Thus, the accessory subunit in 4β2^* AChRs has a large effect on the extent to which cytisine is a partial agonist.

View this table: [in this window] [in a new window]   TABLE 1 4β2* AChRs are more sensitive to block of function by desensitization caused by prolonged exposure to nicotine than to blockage by competitive or noncompetitive antagonists, or than to activation by most agonists

View this table: [in this window] [in a new window]   TABLE 3 Comparison of properties of 4β2 AChR subtypes

In oocytes expressing linked 4 and β2 subunits, the (4β2)₂β3 subtype was found to be approximately as insensitive to ACh as the (4β2)₂4 stoichiometry and similarly high in permeability to Ca²⁺ (Tapia et al., 2007). The 4β2β3 cell line exhibited a biphasic dose/response curve with approximately 18% exhibiting the same high sensitivity to ACh of the (4β2)₂β2 stoichiometry and the remainder exhibiting low sensitivity (Table 1, Fig. 2, D-F). These results suggest that 18% of the surface AChRs in this cell line have the (4β2)₂β2 stoichiometry, whereas more than 80% have the (4β2)₂β3 stoichiometry. Consistent with this, Fig. 1 showed that 13% of the AChRs did not incorporate a β3 subunit. (4β2)₂β3 AChRs were 5-fold less sensitive to nicotine than were (4β2)₂5 AChRs. Cytisine was a 19% agonist on (4β2)₂β3 AChRs expressed in the cell line. (4β2)₂β3 AChRs exhibited 12-fold more nicotine sensitivity than (4β2)₂4 AChRs and 7.8-fold lower sensitivity than (4β2)₂5 AChRs.

Agonists both activate and desensitize AChRs. AChRs in cell lines can be assayed for acute desensitization by agonists and for desensitization over long periods that reflect the time period that they would be exposed to drugs in vivo. 4β2^* AChRs are more sensitive to block of function by desensitization than they are to most competitive or noncompetitive antagonists or than they are to activation by most agonists (Tables 1 and 3). The parent 4β2 line is 14-fold more sensitive to long-term desensitization by nicotine than it is to acute competitive block by DHβE and 126-fold more sensitive to long-term desensitization by nicotine than it is to acute channel block by mecamylamine. Although acute desensitization of the (4β2)₂4 stoichiometry is more rapid (Nelson et al., 2003), both stoichiometries seem to desensitize to the same final state after exposure to nicotine. There is a monotonic long-term desensitization curve to nicotine, indicating that both stoichiometries are equally sensitive to long term desensitization.

(4β2)₂5 AChRs are similarly sensitive to long-term desensitization by nicotine as are AChRs in the parent line (Table 1). (4β2)₂β3 AChRs are 4.5-fold less sensitive to long-term desensitization by nicotine. During exposure to nicotine over 2 min, (4β2)₂5 AChRs desensitize more rapidly to a lower plateau level (15 ± 1%) than do 4β2 (68 ± 9%) AChRs (Fig. 3). These results, assayed using a Ca²⁺-sensitive fluorescent indicator and the FLEXstation, are consistent with results obtained electrophysiologically with (4β2)₂5 AChRs expressed in X. laevis oocytes (Ramirez-Latorre et al., 1996; Kuryatov et al., 1997). (4β2)₂β3 AChRs also acutely desensitize more rapidly than do 4β2 AChRs but less rapidly than (4β2)₂5 AChRs. At the 0.1 to 0.2 µM concentrations of nicotine sustained in the sera of smokers (Benowitz, 1996), the IC₅₀ = 0.009 µM (4β2)₂5 AChRs or the IC₅₀ = 0.027 µM (4β2)₂β3 would result in most of these AChRs being desensitized. Most (4β2)₂5 AChRs would also be desensitized by the 0.0054 µM plasma concentration of nicotine produced by 1 to 2 puffs of a cigarette (Brody et al., 2006).

View larger version (45K): [in this window] [in a new window]   Fig. 3. Acute desensitization of responses to 30 µM ACh assayed using a Ca2+ fluorescent indicator in a FLEXstation. Both (4β2)25 and (4β2)2β3 AChRs desensitize much more rapidly than does the mixture of (4β2)2β2 and (4β2)24 AChRs in the 4β2 cell line. The responses shown are the averages of four microwell cultures from each line.

Galanthamine has been reported to act as a positive allosteric modulator (PAM) of human 4β2 AChRs expressed in permanently transfected HEK 293 cells (Samochocki et al., 2003). We observed that very low concentrations of galanthamine (EC₅₀ = 0.25 nM) increased the response of (4β2)₂5 AChRs to 1 µM ACh by up to 220% (Fig. 4). Only small potentiation (20%) of either 4β2 or (4β2)₂β3 AChRs was detected using FLEXstation assays. Galanthamine at concentrations of 1 µM and above inhibited all three AChR subtypes, consistent with the results of Samochocki et al. (2007). These experiments illustrate the principle that a particular AChR accessory subunit can confer high sensitivity to a PAM.

View larger version (18K): [in this window] [in a new window]   Fig. 4. The PAM galanthamine selectively potentiates (4β2)25 AChRs. ACh and galanthamine were added simultaneously, and activity was assayed using a Ca2+-sensitive indicator in the FLEXstation. ACh was used as agonist at 1 µM for both the 4β2 line and 4β25 line and at 30 µM for the 4β2β3 line in the experiments shown. ACh was also tested on the 4β2 line at 0.1 µM ACh so as to be below the EC50 for the (4β2)2β2 stoichiometry. At this concentration, as at 1 µM, only a small positive allosteric effect was observed. Concentrations of galanthamine below 10 nM (EC50 = 0.25 nM) increase the response of the 4β25 line to an EC50 concentration of ACh by a maximum of 2.2-fold. The responses to near EC50 ACh concentrations of the 4β2 or 4β2β3 cell lines were increased by <20%.

Nicotine up-regulated the amount of (4β2)₂5 AChRs with an EC₅₀ = 0.0353 ± 0.0078 µM and an extent of 4.8-fold (Tables 2 and 3). Thus, 5 does not alter the sensitivity or extent of nicotine-induced up-regulation of 4β2^* AChRs. This might suggest that nicotine acts to promote assembly of 4β2 subunit dimer or 4β24β2 subunit tetramer assembly intermediates before assembly with 5, because 5 decreased sensitivity to activation by nicotine 3-fold and greatly increased sensitivity to rapid desensitization. However, the IC₅₀ for desensitization by nicotine after exposure for hours is approximately the same for the 4β2 and 4β25 lines, and this may be the most relevant parameter for nicotine-induced up-regulation if a desensitized conformation is what promotes assembly. The up-regulated AChRs continued to incorporate 5 efficiently, as shown by the ability of mAb 210 (to 5) to immune precipitate all of the AChRs, which could be immune-precipitated by mAb 295 (to β2 subunits) (Fig. 5).

View this table: [in this window] [in a new window]   TABLE 2 Upregulation of 4β2* AChRs in cell lines

View larger version (18K): [in this window] [in a new window]   Fig. 5. AChRs up-regulated by nicotine still efficiently incorporate 5 subunits. After overnight exposure to the indicated concentrations of nicotine, AChRs in Triton X-100 extracts were immune precipitated by either mAb 295 to measure total AChRs or mAb 210 to measure those that incorporated 5 subunits.

Sensitivity to up-regulation of the 4β25 line by cytisine (EC₅₀ = 0.0058 ± 0.0014 µM) (Table 2) was similar to that of the parent 4β2 cell line (EC₅₀ = 0.0075 ± 0.0027 µM) (Kuryatov et al., 2005). Note that the sensitivities to upregulation (Tables 2 and 3) and desensitization (Table 1) are much greater than the sensitivities to activation (e.g., 3- to 19-fold in the case of nicotine). Note also that agonists are more potent at up-regulation than the competitive antagonist. These results suggest that a desensitized conformation of 4β2 intermediates assembles more efficiently than a resting or active conformation.

By contrast with (4β2)₂5 AChRs, (4β2)₂β3 AChRs were not up-regulated at all by nicotine (Tables 2 and 3). This may be relevant to the observation that the 4β2β3 line expressed AChRs at 25 times the level of the parent 4β2 line, approximately the level obtained when the parental line was maximally up-regulated by nicotine and all of the 4 and β2 subunit pools were incorporated into mature AChRs (Kuryatov et al., 2005), leaving no possibility for a further effect of nicotine. In the case of 6 AChRs, β3 increased the level of expression in cell lines selected from 6β2 or 6β4 parental lines 1.5- to 3.6-fold but also increased the sensitivity to nicotine-induced up-regulation by 6.6- to 11-fold (Tumkosit et al., 2006). In these lines, 6β2 AChRs and 6β4 AChRs were expressed at levels 5% that of the 4β2 line. The presence of β3 resulted in both more mature 6 AChRs and more 6 detected in Western blots, suggesting that 6 was unstable unless incorporated into mature AChRs. By contrast, similar 3β2 and 4β2 cell lines (Wang et al., 1998; Kuryatov et al., 2005) have large stable pools of partially assembled subunits, which can be quickly assembled into many more mature AChRs in the presence of nicotine without requiring the synthesis of new subunits.

Surface Membrane Expression. (4β2)₂β3 AChRs were very efficiently (67 ± 15%) expressed on the cell surface. For comparison, in the 4β2 line, 81% of AChRs are on the surface normally and 60% after up-regulation by nicotine (Kuryatov et al., 2005). Thus, not only are (4β2)₂β3 AChRs efficiently assembled but they are also expressed on the cell surface with high efficiency comparable with the 4β2 cell line. By contrast, (4β2)₂5 AChRs were much less efficiently (20 ± 6%) expressed on the cell surface, and efficiency of expression on the surface was not further increased by up-regulation with nicotine. Specific association of 5 with a postsynaptic scaffold protein may be required for optimal expression on the cell surface (Conroy et al., 2003). These assays measured the fraction of epibatidine binding sites that were expressed on the cell surface. The percentage of mature pentameric AChRs expressed on the cell surface is actually higher than this would indicate because, as will be described later, within the cell nearly half of the epibatidine binding sites are on partially assembled AChRs.

Effects of Chaperones on Incorporation of 5 and β3. A possible explanation for why (4β2)₂β3 AChRs have not been observed in immunoisolation studies from brain (Gotti et al., 2007; Perry et al., 2007; Mao et al., 2008), despite the evidence presented here that β3 can assemble very efficiently with 4β2, is that a specific chaperone present in neurons inhibits the incorporation of β3 on the minus side of 4 subunits. We investigated transient transfection of the 4β25 and 4β2β3 cell lines with Lynx1 (Ibañez-Tallon et al., 2002) and Ric-3 (Lansdell et al., 2005) as candidate chaperones. Neither decreased incorporation of β3 into AChRs (data not shown). Transfection with Lynx1 caused up to a 40% decrease in incorporation of 5 and caused an equal amount of a second low affinity (EC₅₀ = 85.7 µM) component in the 4β25 dose/response curve [probably (4β2)₂4 AChRs]. Transfection with Ric-3 greatly increased expression of mature 7 AChRs in an 7 cell line (data not shown). Thus, Lynx1 and Ric-3 were functional when expressed in tsA201 HEK cell lines, so lack of effect on incorporation of β3 was not due to lack of function of Lynx1 and Ric-3.

Assembly of AChRs Analyzed Using Sucrose Gradient Sedimentation. Partially assembled 4β2 AChRs are disrupted by Triton X-100, and [³H]epibatidine binding is detected to only mature pentameric AChRs on sucrose gradients unless assembly intermediates are stabilized using a cross-linking reagent (Kuryatov et al., 2005). The presence of either 5 or β3 subunits resulted in the formation of partially assembled AChRs, which accounted for approximately half of the total [³H]epibatidine binding sites on the gradients (Fig. 6). These partially assembled AChRs must contain both 4 and β2 to form epibatidine binding sites and must contain accessory subunits to prevent dissociation by Triton X-100. Immunoisolation showed that mature and partially assembled AChRs contained 5 or β3, but the limited affinity of mAb 210 did not allow isolation of all complexes containing 5 or β3 when mAb 210 was used on coated microwells. 4 was immunologically detectable virtually only in mature pentamers, even though 4 had to be present in partially assembled AChRs to permit formation of epibatidine binding sites. This suggests that the 4 in the partially assembled AChRs, especially the large cytoplasmic domain where epitopes recognized by antiserum to 4 and mAb371 are located, was partially proteolytically degraded.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Sucrose gradient sedimentation reveals large amounts of partially assembled AChRs. Cosedimentation on the gradients of Torpedo AChR 9.5S monomers and 13S dimers immunoisolated on mAb210 coated wells and labeled with 125I--bungarotoxin (indicated by arrows) provided internal standards for identifying the 10 S mature pentameric AChRs peaking around fraction 30 and the 8.5 S partially assembled AChRs peaking near fraction 40 after having sedimented from the top of the gradient collected in fraction 60. [3H]epibatidine-labeled AChRs containing β2 subunits were isolated using mAb295-coated microwells. Those containing immunologically recognizable 4 subunits were immunoprecipitated using antiserum to bacterially expressed 4. AChR containing 5 and β3 subunits were isolated on mAb 210 coated microwells.

Up-regulation of 4β25 cells using nicotine resulted in increased amounts of both mature and partially assembled AChRs (Fig. 7). Thus, the molecular chaperone effect of nicotine, which promotes assembly, probably acts on an intermediate containing at least the one 4 and one β2 subunit needed to form an ACh binding site (e.g., 4β2, 54β2, 4β25, 4β24β2) but before formation of dead-end partially assembled AChRs (e.g., 54β25).

View larger version (17K): [in this window] [in a new window]   Fig. 7. Nicotine up-regulates both partially and fully assembled 4β25 AChRs. The positions of the 9.5 S monomer and 13 S dimer of T. californica AChRs on the gradients are shown by arrows.

View larger version (21K): [in this window] [in a new window]   Fig. 8. Transient transfection with additional 4 causes all AChRs to be assembled as mature (4β2)25 AChRs. Sedimentation on sucrose gradients resolved only mature 10 S AChRs. Immunoisolation of [3H]epibatidine labeled AChRs on mAb coated microwells showed that 4, β2, and 5 subunits were present in these AChRs.

The 4β2β3 line also exhibits nearly equal amounts of mature and partially assembled AChRs. In addition, similar to the 4β25 line, all of the epitopes for mAb 371 to the 4 cytoplasmic surface are destroyed (as are epitopes for anti-serum to 4 and mAb 299, which has an extracellular epitope, data not shown) (Fig. 9). Biotinylation of the cell surface with a membrane-impermeable reagent before solubilization permitted identification of AChRs that were on the cell surface by isolation on streptavidin-coated wells. As expected, only mature AChRs were expressed on the cell surface (Fig. 9). Thus, in both the 4β25 and 4β2β3 lines, there are large amounts of partially assembled AChRs stabilized by their accessory subunits. Probably in each case these are formed as a result of large amounts of accessory subunits and limiting amounts of 4 (Fig. 8). In the presence of large amounts of β3 relative to 4, a 4β2β3 assembly intermediate trimer might assemble with β3 to form a stable dead end β34β2β3 complex before it could assemble with 4 then β2 or with an 4β2 pair to form a mature (4β2)₂β3 AChR. The partially assembled AChRs detected on the sucrose gradients are probably partially degraded dead-end complexes of the form β34β2β3. Only mature AChRs get to the cell surface where their function can be assayed.

View larger version (27K): [in this window] [in a new window]   Fig. 9. Sucrose gradient sedimentation analysis of the 4β2β3 line. Microwells coated with mAbs were used to immunoprecipitate [3H]epibatidine-labeled AChRs from gradient fractions revealing that, as with the 4β25 line, there are nearly equal amounts of mature and partially assembled AChRs and that 4 epitopes are detectable in the mature AChRs and not the partially assembled AChRs. The 4β2β3 cells were surface labeled with biotin before solubilization, then streptavidin coated wells were used to isolate AChRs that derived from the cell surface. Only mature AChRs and no partially assembled AChRs were found on the cell surface.

The presence of nearly half of the epibatidine binding sites as intracellular dead end intermediates means that higher proportions of mature AChRs are on the cell surface than was calculated by measurements of the proportion of epibatidine binding sites on the cell surface. Correcting for the amount of binding sites present on intracellular dead end intermediates, virtually all mature (4β2)₂β3 AChRs are expressed on the cell surface as are 34% of mature (4β2)₂5 AChRs.

Obligate Accessory Subunits (5 or β3) and Other Subunits (β4 and 6) Can Rescue Function of ADNFLE 4S247Fβ2 Mutant AChRs. These mutant AChRs do not form functional AChRs in the transfected cell line, presumably because HEK cells preferentially produce (4β2)₂4 AChRs resulting in three phenylalanine groups in the lumen of the channel (Kuryatov et al., 2005).

Displacing the 4 in the accessory position of 4S247Fβ2 mutant AChRs by cotransfection with 5 or β3 results in functional AChRs (Fig. 10). This is consistent with the observation that, when this mutant is expressed in oocytes with subunit ratios that promote assembly of the (4β2)₂β2 stoichiometry, functional AChRs are produced (Kuryatov et al., 1997). β3 was most potent in rescuing function, consistent with its exceptional efficiency in assembling with wild-type AChRs in the 4β2β3 line. β4 subunits could also displace the 4 accessory subunit to form functional AChRs (Fig. 10). They may also have displaced some β2 subunits in forming ACh binding sites, but only displacing the accessory 4 subunits in forming ACh binding sites would reduce the number of phenylalanines blocking the channel. 6 subunits could also rescue function (Fig. 10). These could displace either 4 acting as an accessory subunit or 4 forming part of an ACh binding site. (4β2)(6β2)β3 AChRs have been found in brain (Gotti et al., 2006). In HEK cell lines, it has been difficult, requiring special conditions, to get 4 and 6 to coassemble into AChRs where both 4 and 6 subunits participate in forming ACh binding sites (A.K. and J.L., unpublished observations).

View larger version (38K): [in this window] [in a new window]   Fig. 10. Function of the S247F4β2 cell line AChRs is rescued by transient transfection with 5, β3, β4, or 6 subunits. After transfection overnight with the indicated subunits, function was assayed using the FLEXstation with a Ca2+-sensitive indicator. The strongest responses to application of ACh are shown.

View this table: [in this window] [in a new window]   TABLE 4 Activation of S247F4β2* AChRs in a cell line

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The obligate accessory subunits 5 and β3 are efficiently incorporated with human 4 and β2 AChR subunits expressed in permanently transfected HEK cell lines, thereby revealing properties of (4β2)₂5 and (4β2)₂β3 AChRs that are likely to be relevant to the expression of these subtypes in neurons. Expression of excess accessory subunits resulted in their incorporation in nearly all of the AChRs in these cell lines.

(4β2)₂5 AChRs are known to be expressed in brain and elsewhere (Gotti et al., 2007). We have shown that 5 increased Ca²⁺ permeability compared with (4β2)₂β2 AChRs and sensitivity to activation compared with (4β2)₂4 AChRs (Tapia et al., 2007), and that 5 restored Ca²⁺ permeability to (S247F4β2)₂β2 AChRs (Kuryatov et al., 1997). The 4β25 line expressed 40% more AChRs than the parental 4β2 line. This permitted further up-regulation of expression by nicotine and other agonists with the same sensitivity as the 4β2 line. 5 increased sensitivity to activation by agonists compared with (4β2)₂4 AChRs, increased the rate of acute desensitization by nicotine, but did not change sensitivity to long-term desensitization.

(4β2)₂β3 AChRs are not usually considered among brain ACh subtypes, but because 4, β2, β3, and 6 subunits are all assembled in the endoplasmic reticulum of dopaminergic neurons like those of the ventral tegmental area or the substantia nigra, which are known to assemble (6β2)(4β2)β3 AChRs (Gotti et al., 2007), there is ample opportunity for their synthesis. In the transfected line, (4β2)₂β3 AChRs assemble with exceptional efficiency so that very high levels of AChRs are expressed on the cell surface. No further upregulation by nicotine was observed, probably because 4 and β2 subunit pools were depleted.

The stoichiometry and subunit composition of AChRs expressed in neurons may depend critically on transcriptional or translational regulation of subunit synthesis determining the pools of subunits available for assembly. In X. laevis oocytes, injecting an excess of 4 mRNA results in the (4β2)₂4 stoichiometry, whereas injecting an excess of β2 mRNA results in the (4β2)₂β2 stoichiometry (Moroni et al., 2006).

Immunoisolation studies from rat brains indicate that the amount of 4 and β2 subunits always greatly exceeds the amount of 5 subunits (Gotti et al., 2007; Perry et al., 2007; Mao et al., 2008). This accounts for the observation that (4β2)₂5 AChRs are only 11 to 37% of the total 4β2^* AChRs, depending on the brain region (Mao et al., 2008). Long-term exposure to nicotine increases the amount of brain 4β2^* AChRs but not the amount of (4β2)₂5 AChRs (Mao et al., 2008). This might result from the synthesizing of 5 in limiting amount, all of which is assembled, leaving only pools of 4 and β2 to be assembled in response to the pharmacological chaperone effects of nicotine. We show here that when 5 is present in excess, it assembles efficiently with 4β2 and the amount of 4β25 AChRs is increased in the presence of nicotine. In rat brain, less β3 is expressed than 5, and the amounts of 4, β2, 5, and 6 exceed the amount of β3 (Gotti et al., 2007; Perry et al., 2007; Mao et al., 2008). Furthermore, 5 is always found in association with 4, and β3 is always in association with 6, never with 4 alone. As with 5, after up-regulation by nicotine, the amount of β3 remains constant, indicating that all of the limited amount of β3 is already incorporated in AChRs. Here we show that β3 can assemble efficiently with 4β2. The absence of (4β2)₂β3 AChRs in brain may result from a combination of the limiting amount of β3 and perhaps also greater affinity of β3 for assembling with 6 than 4 and greater affinity of 5 for assembling with 4 than 6.

We show that when 5 and β3 are expressed in amounts equal to or greater than 4 and β2, many dead-end, partially assembled AChRs are formed. Thus, the observation that only small amounts of 5 and β3 are usually expressed may reflect a biological necessity to avoid forming nonproductive assemblies. Both β34β2 and 4β2β3 subunit trimers have allowable subunit interfaces and can form mature AChRs by assembly with 4β2 dimers. With excess β3, β34β2β3 tetramers are likely to form, which have allowable interfaces but cannot form a mature pentamer with addition of another 4 or β2. The stability of putative β34β2β3 tetramers to dissociation by Triton X-100 and the proteolytic decay of the 4 within them indicate that they are not easily eliminated by conventional editing mechanisms and are thus potentially toxic.

Demonstration in cell lines that various AChR subunits can assemble efficiently and be up-regulated by nicotine, in combination with the concept that in neurons some subunits are synthesized in limiting amounts, can explain several conundrums. After treatment of rats with nicotine for 2 weeks, in the striatum 4β2^* AChRs are increased (as measured by ligand binding), 6β2^* AChRs are decreased, and the total amount of β3-containing AChRs remains constant (Perry et al., 2007). These results might be explained if 4β2 AChRs in GABAergic neurons were up-regulated and the total amount of 4 subunit in dopaminergic neurons remained constant (Nashmi et al., 2007), but in the dopaminergic neurons, 6 was displaced by 4 from (6β2)(4β2)β3 AChRs to form (4β2)₂β3 AChRs. This would be expected if nicotine acted on 4β2 and 6β2 subunit pairs to promote assembly because nicotine is much more potent at promoting the assembly of 4β2 than 6β2 AChRs (Kuryatov et al., 2005; Tumkosit et al., 2006). We show here that β3 avidly assembles with 4β2 and in Tumkosit et al. (2006) that it avidly assembles with 6β2; thus, all the β3 present will assemble. In rat superior colliculus, nicotine did not reduce the numbers of 6^* AChRs (Perry et al., 2007). This would be expected if the retinal ganglia neurons that terminate in the superior colliculus expressed only (6β2)₂β3 AChRs and did not also express 4 to compete for assembly, as do ventral tegmental area dopaminergic neurons.

Both obligate accessory subunits (5 and β3) and other subunits (β4 and 6) can restore function to the ADNFLE mutant cell line S247F4β2. Displacing the 4 in the accessory position to form, for example, (S247F4β2)₂β3 AChRs leaves only two phenylalanines in the cation channel. Remarkably, this not only unblocks the channel but also greatly increases sensitivity to activation through some interaction between the phenylalanine groups and the β3 subunit. There is precedent for mutations in the M2 region of 4 and other subunits greatly increasing sensitivity to activation (Labarca et al., 2001).

As shown here and elsewhere, 5 and β3 subunits can have substantial effects on the efficiency of assembly of the AChR subtypes that contain them and on the pharmacological, conductance, and desensitization properties of these AChRs. In addition, 5 and β3 subunits may have important roles in targeting AChRs to particular locations (Gotti et al., 2007). In the ventral tegmental area, dopaminergic neurons, 6^* AChRs containing β3 subunits, are selectively located at presynaptic endings (Champtiaux et al., 2003; Quik et al., 2007). In transfected N2a cells, 4β2 AChRs are localized to filopodia, but (4β2)₂β3 AChRs are not (Drenan et al., 2008). When expressed in HEK cells, 5 and 3 (but not 4 and β2) specifically associate with PSD-93a, PSD-95, and SAP102, proteins that are associated with postsynaptic densities (Conroy et al., 2003). Disrupting interactions with postsynaptic density proteins in neurons expressing (3β4)₂5 AChRs impairs excitatory postsynaptic currents without altering the number of AChRs by disrupting alignment of pre- and postsynaptic elements. It seems likely that these accessory subunits could target (4β2)₂5 and (4β2)₂β3 AChRs to particular post- and presynaptic locations.

GABA_A receptors are homologous in structure to nicotinic AChRs. In these receptors, the presence of the accessory subunit is necessary to permit formation of a binding site for benzodiazepines at the interface between and subunits (Cromer et al., 2002). PAMs such as benzodiazepines have great theoretical significance as drugs because they can promote the effectiveness of neurotransmission without altering the pattern of signaling and can sustain their effects, unlike competitive agonists, which may act as time-averaged antagonists through desensitizing the receptors. Potent PAMs have been reported for 7 AChRs (Hurst et al., 2005; Ng et al., 2007). Galanthamine binds at a subunit interface similar to that at which benzodiazepines bind (Hansen and Taylor, 2007) and acts as a PAM on AChRs (Samochocki et al., 2003). We showed that (4β2)₂5, unlike 4β2 or (4β2)₂β3 AChRs, is uniquely sensitive to galanthamine. Unique interfaces, like those between 5 and 4 or between β3 and 6, could provide very specific drug targets for PAMs more easily discriminated than ACh binding sites for selectively targeting AChR subtypes. Cell lines like those described here could be critically important in screening for such positive allosteric modulators.

	Acknowledgements

 
We thank Barbara Campling for comments on the manuscript.

	Footnotes

 
This work was supported by grant NS11323 from the National Institutes of Health (to J.L.).

ABBREVIATIONS: AChR, acetylcholine receptor; ACh, acetylcholine; HEK, human embryonic kidney; ADNFLE, autosomal-dominant nocturnal frontal lobe epilepsy; PBS, phosphate-buffered saline; mAb, monoclonal antibody; PAM, positive allosteric modulator; DHβE, dihydro-β-erythroidine.

Address correspondence to: Jon Lindstrom, Department of Neuroscience, University of Pennsylvania Medical School, 217 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104. E-mail: jslkk{at}mail.med.upenn.edu

	References

Top Abstract Materials and Methods Results Discussion References

 
Benowitz N (1996) Pharmacology of nicotine: addiction and therapeutics. Annu Rev Pharmacol Toxicol 36: 597-613.[CrossRef][Medline]

Briggs CA, Gubbins EJ, Marks MJ, Putman CB, Thimmapaya R, Meyer MD, and Surowy CS (2006) Untranslated region-dependent exclusive expression of high-sensitivity subforms of 4β2 and 3β2 nicotinic acetylcholine receptors. Mol Pharmacol 70: 227-240.[Abstract/Free Full Text]

Brody AL, Mandelkern MA, London ED, Olmstead RE, Farahi J, Scheibal D, Jou J, Allen V, Tiongson E, Chefer SI, et al. (2006) Cigarette smoking saturates brain alpha 4 beta 2 nicotinic acetylcholine receptors. Arch Gen Psychiatry 63: 907-915.[Abstract/Free Full Text]

Brown R, Collins AC, Lindstrom J, and Whiteaker P (2007) Nicotinic 5 subunit deletion locally reduces high affinity agonist activation without altering receptor numbers. J Neurochem 103: 204-215.[Medline]

Champtiaux N, Gotti C, Cordero-Erausquin M, David D, Przbyloki C, Lena C, Clementi F, Moretti M, Rossi F, LeNovere N, et al. (2003) Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knockout mice. J Neurosci 23: 7820-7829.[Abstract/Free Full Text]

Conroy WG, Liu Z, Nai Q, Coggan JS, and Berg DK (2003) PDZ-containing proteins provide a functional postsynaptic scaffold for nicotinic receptors in neurons. Neuron 38: 759-771.[CrossRef][Medline]

Cromer BA, Morton CJ, and Parker MW (2002) Anxiety over GABA(A) receptor structure relieved by AChBP. Trends Biochem Sci 27: 280-287.[CrossRef][Medline]

Drenan RM, Nashmi R, Imoukhuede PI, Just H, McKinney S, and Lester HA (2008) Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled 6 and β3 subunits. Mol Pharmacol 73: 27-41.[Abstract/Free Full Text]

Gerzanich V, Wang F, Kuryatov A, and Lindstrom J (1998) 5 Subunit alters desensitization, pharmacology, Ca²⁺ permeability and Ca²⁺ modulation of human neuronal 3 nicotinic receptors. J Pharmacol Exp Ther 286: 311-320.[Abstract/Free Full Text]

Gotti C, Zoli M, and Clementi F (2006) Brain nicotinic acetylcholine receptors: native subtypes and their relevance. Trends Pharmacol Sci 27: 482-491.[CrossRef][Medline]

Gotti C, Moretti M, Gaimarri A, Zanardi A, Clementi F, and Zoli M (2007) Heterogeneity and complexity of native brain nicotinic receptors. Biochem Pharmacol 74: 1102-1111.[CrossRef][Medline]

Hansen SB and Taylor P (2007) Galanthamine and non-competitive inhibitor binding to ACh-binding protein: evidence for a binding site on non-alpha-subunit interfaces of heteromeric neuronal nicotinic receptors. J Mol Biol 369: 895-901.[CrossRef][Medline]

Hurst RS, Hajos M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-Root KL, Berkenpas MB, Hoffmann WE, Piotrowski DW, et al. (2005) A novel positive allosteric modulator of the 7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J Neurosci 25: 4396-4405.[Abstract/Free Full Text]

Ibañez-Tallon I, Miwa J, Wang HL, Adams N, Crabtree G, Since S, and Heintz N (2002) Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1. Neuron 33: 893-903.[CrossRef][Medline]

Kedmi M, Beaudet AL, and Orr-Urtreger A (2004) Mice lacking neuronal nicotinic acetylcholine receptor beta4-subunit and mice lacking both alpha5- and beta4-subunits are highly resistant to nicotine-induced seizures. Physiol Genomics 17: 221-229.[Abstract/Free Full Text]

Klaassen A, Glykys J, Maguire J, Labarca C, Mody I, and Boulter J (2006) Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy. Proc Natl Acad Sci U S A 103: 19152-19257.[Abstract/Free Full Text]

Kuryatov A, Gerzanich V, Nelson M, Olale F, and Lindstrom J (1997) Mutation causing autosomal dominant nocturnal frontal lobe epilepsy alters Ca²⁺ permeability, conductance, and gating of human 4β2 nicotinic acetylcholine receptors. J Neurosci 17: 9035-9047.[Abstract/Free Full Text]

Kuryatov A, Olale F, Cooper J, Choi C, and Lindstrom J (2000) Human 6 AChR subtypes: subunit composition, assembly, and pharmacological responses. Neuropharmacology 39: 2570-2590.[CrossRef][Medline]

Kuryatov A, Luo J, Cooper J, and Lindstrom J (2005) Nicotine acts as a pharmacological chaperone to upregulate human 4β2 acetylcholine receptors. Mol Pharmacol 68: 1839-1851.[Abstract/Free Full Text]

Labarca C, Schwarz J, Deshpande P, Schwarz S, Nowak MW, Fonck C, Nashmi R, Kofuji P, Dang H, Shi W, et al. (2001) Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety. Proc Natl Acad Sci U S A 98: 2786-2791.[Abstract/Free Full Text]

Lansdell SJ, Gee VJ, Harkness PC, Doward AI, Baker ER, Gibb AJ, Millar NS (2005) RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol Pharmacol 68: 1431-1438.[Abstract/Free Full Text]

Lindstrom J (2000) The structure of neuronal nicotinic receptors. Handb Exp Pharmacol (144): 101-162.

Mao D, Perry DC, Yasuda RP, Wolfe BB, and Kellar KJ (2008) The 4β25 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo. J Neurochem 104: 446-456.[Medline]

Marks MJ, Meinerz NM, Drago J, and Collins AC (2007) Gene targeting demonstrates that 4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [³H]epibatidine binding sites and components of biphasic ⁸⁶Rb⁺ efflux with high and low sensitivity to stimulation by acetylcholine. Neuropharmacology 53: 390-405.[CrossRef][Medline]

Moroni M, Zwart R, Sher E, Cassels B, and Bermudez I (2006) 4β2 Nicotinic receptors with high and low acetylcholine sensitivity: pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine. Mol Pharmacol 70: 755-768.[Abstract/Free Full Text]

Nashmi R, Xiao C, Deshpande P, McKinney S, Grady SR, Whiteaker P, Huang Q, McClure-Begley T, Lindstrom JM, Labarca C, et al. (2007) Chronic nicotine cell specifically upregulates functional alpha 4^* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path. J Neurosci 27: 8202-8218.[Abstract/Free Full Text]

Nelson M, Kuryatov A, Choi C, Zhou Y, and Lindstrom J (2003) Alternate stoichiometries of 4β2 nicotinic acetylcholine receptors. Mol Pharmacol 63: 332-341.[Abstract/Free Full Text]

Ng HJ, Whittemore ER, Tran MB, Hogenkamp DJ, Broide RS, Johnstone TB, Zheng L, Stevens KE, and Gee KW (2007) Nootropic 7 nicotinic receptor allosteric modulator derived from GABA_A receptor modulators. Proc Natl Acad Sci U S A 104: 8059-8064.[Abstract/Free Full Text]

Perry DC, Mao D, Gold AB, McIntosh JM, Pezzullo JC, and Kellar KJ (2007) Chronic nicotine differentially regulates 6- and β3-containing nicotinic cholinergic receptors in rat brain. J Pharmacol Exp Ther 322: 306-315.[Abstract/Free Full Text]

Quik M, O'Neill M, and Perez XA (2007) Nicotine neuroprotection against nigrostriatal damage: importance of the animal model. Trends Pharmacol Sci 28: 229-235.[CrossRef][Medline]

Ramirez-Latorre J, Yu CR, Qu X. Perin F, Karlin A, Role L (1996) Functional contributions of 5 subunit to neuronal acetylcholine receptor channels. Nature 380: 347-351.[CrossRef][Medline]

Salas R, Orr-Urtreger A, Broide R, Beaudet A, Paylor R, and DeBiasi M (2003) The nicotinic acetylcholine receptor subunit 5 mediates short-term effects of nicotine in vivo. Mol Pharmacol 63: 1059-1066.[Abstract/Free Full Text]

Sallette J, Pons S, Devillers-Thiery A, Soudant M, Carvalho LP, Changeux JP, and Corringer PJ (2005) Nicotine upregulates its own receptors through enhanced intracellular maturation. Neuron 46: 595-607.[CrossRef][Medline]

Salminen O, Drapeau JA, McIntosh JM, Collins AC, Marks MJ, and Grady SR (2007) Pharmacology of -conotoxin MII-sensitive subtypes of nicotinic acetylcholine receptors isolated by breeding of null mutant mice. Mol Pharmacol 71: 1563-1571.[Abstract/Free Full Text]

Samochocki M, Höffle A, Fehrenbacher A, Jostock R, Ludwig J, Christner C, Radina M, Zerlin M, Ullmer C, Pereira EFR, et al. (2003) Galantamine is an allosterically potentiating ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors. J Pharmacol Exp Ther 305: 1024-1036.[Abstract/Free Full Text]

Tapia L, Kuryatov A, and Lindstrom J (2007) Ca²⁺ permeability of the 4₃β2₂ stoichiometry greatly exceeds that of 4₂β2₃ human AChRs. Mol Pharmacol 71: 769-776.[Abstract/Free Full Text]

Teper Y, Whyte D, Cahir E, Lester HA, Grady SR, Marks MJ, Cohen BN, Fonck C, McClure-Begley T, McIntosh JM, et al. (2007) Nicotine-induced dystonic arousal complex in a mouse line harboring a human autosomal-dominant nocturnal frontal lobe epilepsy mutation. J Neurosci 27: 10128-10142.[Abstract/Free Full Text]

Tumkosit P, Kuryatov A, Luo J, and Lindstrom J (2006) β3 subunits promote expression and nicotine-induced up-regulation of human nicotinic 6^* AChRs expressed in transfected cell lines. Mol Pharmacol 70: 1358-1368.[Abstract/Free Full Text]

Wang F, Nelson M, Kuryatov A, Keyser K, and Lindstrom J (1998) Chronic nicotine treatment upregulates human 3β2, but not 3β4 AChRs stably transfected in human embryonic kidney cells. J Biol Chem 273: 28721-28732.[Abstract/Free Full Text]

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