Correspondence to: Jon Lindstrom, 217 Stemmler Hall, University of Pennsylvania Medical School, Philadelphia, PA 19104-6074. Fax:(215) 573-2015 E-mail:jslkk{at}mail.med.upenn.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human 3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express
3,
5,
7, ß2, and ß4 AChR subunits. From these subunits, several types of
3 AChRs as well as homomeric
7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect
3ß4 AChRs.
7 AChR function was not detected, yet we estimate that there are 70% as many surface
7 AChRs in IMR-32 when compared with
3 AChRs. Agonist potencies (EC50 values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16±1 µM) > nicotine (Nic; 48 ± 7 µM)
cytisine (Cyt; 57 ± 3 µM) = acetylcholine (ACh; 59 ± 6 µM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s-1 (300 µM ACh; -60 mV). Assays that used mAbs confirmed the predominance of
3- and ß4-containing AChRs in IMR-32 cells. Although 18% of total
3 AChRs contained ß2 subunits, no ß2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface
3ß2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in
3ß2 transfected HEK cells. Characterization of various
3 AChRs expressed in HEK cell lines revealed that the functional properties of the
3ß4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC50 values) for this line was DMPP (14 ± 1 µM) = Cyt (18 ± 1 µM) > Nic (56 ± 15 µM > ACh (79 ± 8 µM). The efficacies of both Cyt and DMPP were
80% when compared with ACh and the desensitization rate was 2 s-1. These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to
3ß4 AChRs.
Key Words: nicotinic receptor, autonomic, patch clamp
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
3 acetylcholine receptors (AChRs)* have been identified as the predominant neuronal AChR in ganglia of the autonomic nervous system (
3,
5,
7, ß2, and ß4. Much evidence suggests that the
3 AChR is the main mediator of the nicotinic postsynaptic response in autonomic ganglia (
3 subunits illustrate the critical role played by these AChRs in the peripheral nervous system. These animals have increased perinatal mortality and exhibit severe physiologic impairment in organs innervated by the autonomic nervous system (
7 AChRs are expressed widely in ganglia, also. Selective inhibition of
3 AChRs failed to block synaptic transmission in chick ciliary ganglion showing that
7 AChRs are sufficient to sustain transmission in this preparation (
7 AChRs had normal nervous system development (
3 function in mice is lethal largely due to autonomic dysfunction, the inability of
7 AChRs to sustain adequate function brings into question its specialized role in the autonomic nervous system.
Knockout mice that lack both ß2 and ß4 subunits are similar in phenotype to the 3 knockout animals (
3ß2,
3ß4, or
7 AChRs normally contribute to ganglionic transmission, and what other roles do they have? A better understanding of the functional properties of these AChRs as well as how AChR expression might be regulated will help to clarify these issues. IMR-32 cells provide a useful model with which to study the subunit composition of human ganglionic AChRs as they express the characteristic complement of "ganglionic" AChR subunits. Additionally, we have developed a series of cell lines that express
3 AChRs formed from combinations of these ganglionic subunits. We determined and then compared the functional properties of recombinant AChRs with "native" AChRs that are expressed by IMR-32 cells. We use functional and pharmacological profiling to investigate further the subunit composition of human AChRs expressed by IMR-32 cells and support our conclusions with various antibody-based molecular techniques.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Culture
Transfected human embryonic kidney (HEK) tsA201 cells were maintained in DME medium as described previously (3 subunit expression, 0.6 mg/ml G-418 (Life Technologies) was used for ß2 or ß4 subunit selection, and 0.2 mg/ml hygromycin B (Boehringer Mannheim) was used for
5 subunit selection. IMR-32 neuroblastoma cells (
Electrophysiology
At least 2 d before recording, HEK cells were plated onto glass coverslips coated with rat tail collagen (Type 1; Collaborative Biomedical Products), and the IMR-32 cells were plated onto glass coverslips coated with mouse laminin (Collaborative Biomedical Products). Currents were measured by standard patch-clamp techniques () were formed from borosilicate glass and were filled with a solution containing the following (in mM): 150 cesium gluconate, 10 Cs-EGTA, and 10 HEPES, adjusted to pH 7.2 with CsOH. Cell access resistances were typically 815 M
and were compensated (4060%) when peak currents were in excess of 2 nA. Cells transfected with
3ß2 or
3
5ß2 AChRs were treated for 12 h with Nic (100 µM) followed by a minimum of 1-h wash with normal media before recording (
where y is the normalized response amplitude, A1 is the maximum amplitude asymptote of the fit, A2 is the minimum asymptote of the fit, x is the concentration of agonist, x0 is the EC50 value, and p is the steepness of the fitted curve. In some cases, the concentrationresponse relationship would peak and then decline with increasing concentrations of agonist. In the cases where the decline caused an obviously inferior fit, the reduced amplitude responses at higher concentrations were not included in the fit. Desensitization time constants were determined by fitting exponential equations to the data. Representative traces were constructed by opening data files in Axograph 3.55 (Axon Instruments) and exporting data to Canvas 5.0 (Deneba Software, Inc.).
Single-Channel Analysis
Single-channel currents were recorded and analyzed as described previously (
Production of mAb 337
mAb 337 (mouse IgG) was developed from a bacterially expressed fusion protein consisting of the large cytoplasmic loop located between the M3 and M4 transmembrane domains of the human ß4 subunit (amino acids 305419) coupled at the NH2 terminus to bacterial glutathione S-transferase (3ß4 AChRs (from transfected HEK cells) in immunoprecipitation assays. 5 d after a final boost with antigen, the animal with the highest titers was killed. Hybridomas for mAb production were formed by fusion of the animal's splenic lymphocytes with Sp2 myeloma cells according to a procedure adapted from (
3ß4 AChRs by immunoprecipitation assays and positive wells were cloned. As we show here, in addition to recognizing the ß4 subunit in its native confirmation, the antibody also recognizes the denatured subunit as well. Lack of cross-reactivity with human
3,
5, or ß2 subunits was established using AChRs extracted from the
3
5ß2 cell line with mAb 337-coated microwells using the procedure described below for radioimmune assays. All animals were handled in accordance with guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania under approved protocol on file with that office. IACUC operates under an institutional Animal Welfare Assurance (A3079-01) on file with the Office for Protection from Research Risks at the National Institutes of Health.
Radioimmune Assays (RIAs)
IMR-32 and SH-SY5Y cells were grown as described above until just before confluence in T-175 (175 cm2) tissue culture flasks (model Falcon 3112; Becton Dickinson) then detached with 5 mM EDTA in PBS, pelleted by centrifugation, and used immediately or frozen after removal of media. Fresh or frozen IMR-32 cells were suspended and triturated in a 2% Triton X-100 in buffer A (50 mM Na2HPO4-NaH2PO4, pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride at a 3:16:1 ratio (buffer volume/cell volume) and placed on a rotating mixer for 23 h at 4°C. Cellular debris was removed by centrifugation and supernatant extract was collected.
For solid phase RIA, AChRs solubilized in Triton were incubated in Immulon 4 microwells (Dynatech Laboratories) coated with mAb 210 (specific for 3 and
5 AChRs;
To measure the amount of 3-containing AChRs that could be labeled in intact cells by [3H]epibatidine, IMR-32 cells were plated in 35-mm tissue culture dishes and grown until confluent. At this time, the media was replaced with 0.5 ml of media that contained 2 nM [3H]epibatidine and returned to the tissue culture incubator. For background determinations, 300 µM Nic was added 45 min before [3H]epibatidine. After 2 h at 37°C, the medium was then removed and the cells quickly rinsed with PBS. The cells were removed from the dishes by trituration with 0.5 ml buffer A, and then lysed with an ultrasonic cell disrupter (model Sonifier 200; Branson Ultrasonics Corp.). Membrane fragments were collected on glass fiber filters (model GF/F; Whatman; 25 mm, treated with 0.3% polyethylenimine) followed by four rapid 1-ml washes with ice cold PBS. Filters were then transferred to scintillation tubes for counting of retained [3H]epibatidine.
For isolation and measurement of 7-containing AChRs, microwells were coated with a combination of mAbs 306 and 319 that recognizes human
7 subunits (
-BGT (20 nM; 0.48 Ci/µmol). For background determinations, unlabeled
-BGT (200 nM) was included in the incubation 1 h before addition of 125I-
-BGT. After overnight incubation, unbound material was removed as described above and the amount of bound 125I-
-BGT was determined by gamma counting.
Surface AChR Labeling
To measure surface AChRs, cells were collected from tissue culture flasks and gently pelleted by centrifugation followed by a wash in normal culture medium containing 10% FBS. Cells were resuspended in tissue culture medium and aliquoted (500 µl) into microcentrifuge tubes. Surface 3-containing AChRs were labeled by incubation (24 h) with 125I-mAb 210 (4 nM; 0.46 Ci/µmol) and surface ß2-containing AChRs were labeled with 125I-mAb 295 (46 nM; 0.69 Ci/µmol). Background determinations were made in parallel by including at least 50-fold excess unlabeled mAb in a 1 h preincubation to block specific sites. Incubations were carried out at 37°C with constant mixing. After incubation, cells were diluted with an equal volume of tissue culture media, pelleted, and then washed twice more with clean media. Amount of bound 125I-mAb was then determined by gamma counting.
Western Blotting
Triton X-100 extraction of AChRs from IMR-32 and SH-SY5Y neuroblastoma cells were performed as described above. The extracts were incubated overnight at 4°C with mAb-coupled agarose to select and concentrate AChRs. AChRs were isolated using activated CH Sepharose 4B resin (Amersham Pharmacia Biotech) coupled with mAb 210 for 3 AChRs, coupled with mAb 295 for ß2 AChRs, or coupled with mAb 337 for ß4 AChRs (all at 2 mg mAb/ml of resin). After incubation, bound AChRs were eluted using 3% SDS sample buffer without reducing agents. The samples were loaded on 12% polyacrylamide gels containing SDS and electrophoresed. Transfer of the proteins was done in a semi-dry electroblotting chamber (Semi-Phor; Hoefer Scientific Instruments) to Trans-Blot® medium PVDF membrane (Bio-Rad). The blot membranes were then quenched for 1 h at room temperature with 5% dried milk in PBS containing 0.5% Tween 20 and 10 mM NaN3 (PBS/Tween) with constant agitation. Antisera against
3 or ß2 subunits (
Immunofluorescence with Confocal Microscopy
IMR-32 or permanently transfected cells were plated on either laminin-coated or collagen-coated glass coverslips, respectively, and fixed with 10% formalin/PBS for 1 h at room temperature for labeling with fluorescent antibody to track the presence of AChR subunit proteins. Fixed cells were then washed three times with PBS to remove fixative. Attachment of fluorophores to primary antibodies was done using a kit (Molecular Probes) to attach either Alexa fluor 488 or 594 to the antibodies. Primary labeling with unlabeled antibody was performed overnight at 4°C in PBS containing antibody with 5% goat serum. Secondary labeling was carried out after washing three times with PBS to remove the unbound primary antibody, followed by Alexa fluor 594-labeled GART (Molecular Probes) for mAb 295 or Alexa fluor 568-labeled goat antimouse (GAM) IgG (Molecular Probes) for mAb 337. Secondary antibodies were used at final concentrations of 4 µg/ml for GART or 3 µg/ml for GAM, and were applied for 34 h at room temperature in the presence of 5% goat serum. After washing three times with PBS, primary labeled mAb 210 (Alexa fluor 488) was applied overnight at 4°C at a final concentration of 5 µg/ml in the presence of 5% normal rat serum. To access intracellular AChR subunits, cells were permeabilized with 0.5% Triton X-100 for 1 h at room temperature before labeling with antibodies. To-Pro 3 iodide (Molecular Probes) was included as a nuclear counterstain at 0.51 µM for 2060 min. Coverslips with labeled cells were rinsed twice with PBS and once with deionized water, then mounted on slides using Pro-Long antifade (Molecular Probes), dried, and stored at 4°C until viewed. Images of labeled cells were then acquired using a Leica TCS 4 D laser scanning confocal microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pharmacological Properties of Functional AChRs in IMR-32 Cells
Rapid application of nicotinic agonists to patch-clamped IMR-32 cells evoked responses ranging up to several hundred picoamps when clamped at -60 mV. An example of a concentration/response family of currents from a cell is shown in Fig 1A using Cyt. The concentration/response relationships are shown for ACh, Nic, DMPP, and Cyt along with the curve fits used to determine the EC50 values (Fig 1 B). The rank order of potency was DMPP > Nic Cyt = ACh. This rank order provided a profile for functional native AChR(s) present in IMR-32 cells for comparison with agonist profiles for AChRs of defined composition expressed in transfected cells as described below.
|
All of the agonists exhibited nearly full efficacy (i.e., 80%) with respect to ACh. The maximum responses to ACh and Cyt are shown for the cell in Fig 1 A to illustrate the efficacy of Cyt, which was similar to that of Nic and slightly greater than the efficacy of DMPP. At high agonist concentrations, "rebound" currents were frequently observed, and the peak current amplitudes were often reduced compared with lower agonist concentrations. This was especially true for DMPP. This effect was likely the result of channel block by the agonist and the rebound current occurred as the channel block was relieved on washout.
Functional Properties of IMR-32 AChRs
The current-voltage relationship for ACh-activated currents from IMR-32 cells were studied between -120 and +80 mV (Fig 1 C). The currents approached reversal when the holding potential was between -10 and 0 mV, which is expected for nonselective cation channels under these recording conditions. Additionally, strong inward rectification was found at positive holding potentials until +60 mV, where some reversal was observed.
Desensitization of currents recorded from IMR-32 cells was usually best described by a single exponential function which could be fit with a time constant of decay of = 368 ± 94 ms (n = 8) (Fig 1 D). In some cases, the desensitization was fit best by a double exponential function with time constants of 73 ± 27 ms and 550 ± 136 ms (n = 2). In addition to desensitization during the application of agonist, the peak amplitude of the currents decreased slightly over the course of a full concentration/response experiment. This "rundown" was not prevented by inclusion of various agents in the recording electrode such as ATP, creatine phosphokinase, phosphocreatine, and magnesium, or various combinations of these substances. Additionally, using a recording solution that was based on CsCl also did not prevent the rundown (unpublished data).
Effects of Nicotinic Antagonists on IMR-32 Currents
Responses to 100 µM ACh were antagonized by coapplication of the antagonists d-tubocurare, mecamylamine, and hexamethonium with IC50 values of 0.4 ± 0.2, 3.2 ± 0.6, and 8.5 ± 3.0 µM, respectively (Fig 2 A). The reversible 7 selective antagonist, methyllycaconitine (MLA), had no effect on Nic-activated (300 µM) currents when applied at 20 nM with a 10 min preincubation (Fig 2 B). This concentration of MLA (20 nM) was almost three orders of magnitude higher than the IC50 reported for homomeric
7 AChRs (
7 containing AChRs in rat hippocampal neurons (
7 AChRs. To validate the ability of our application system to activate
7 AChRs efficiently before they desensitized, we recorded the rapidly desensitizing Nic-activated currents that have been attributed to
7 AChRs from rat hippocampal neurons (unpublished data). Thus, it was concluded that any contribution of
7 AChRs to macroscopic currents from IMR-32 cells was negligible.
|
Agonist Potencies and Efficacies in Permanently Transfected 3 AChR Cell Lines
As reported previously (3,
5, ß2, and ß4 subunits) responded robustly to nicotinic agonists. Peak currents ranged up to 15 nA when clamped at -60 mV. Here, we present a more thorough functional characterization of these cell lines so that we can compare, under like conditions, the properties of AChRs with known subunit composition to the properties IMR-32 AChRs as described above. Examples of currents activated by ACh (300 µM) or Cyt (300 µM) from each cell type (
3ß4,
3ß2,
3ß4
5, and
3ß2
5) are shown in Fig 3. For
3ß4 transfected cells, the agonist potency rank order was DMPP = Cyt > Nic > ACh, with EC50 values ranging from 15 µM for DMPP to 79 µM for ACh (Fig 4). Cyt exhibited 60% efficacy, whereas DMPP had 50% efficacy and Nic had
85% efficacy (Fig 4).
|
|
The 3ß2 and
3
5ß2 cell lines expressed few surface AChRs unless the cells were incubated in Nic (
3ß2 AChRs was markedly different from that for
3ß4 AChRs, with the rank order of potency DMPP >> Cyt
Nic >> ACh, which reflected EC50 values that ranged from 9 µM for DMPP to 209 µM for ACh. Consistent with previous studies of
3ß2 AChRs (
3ß2 AChRs (Fig 3 and Fig 4). This was in contrast to the much higher efficacy of Cyt on the
3ß4 (75%),
3ß4
5 (75%), or IMR-32 (80%) cell lines. In addition, DMPP exhibited efficacy greater than ACh. Thus, agonist efficacies for Cyt and DMPP relative to ACh are useful in predicting the presence of the ß2 subunit in native
3 AChRs.
Reduced Temperature Increases Functional 3ß2 AChRs
When incubated at reduced temperature (29°C) overnight, 3ß2 cells increased their amount of functional AChRs as judged by the gain of responsiveness to agonist, much like when the cells were incubated in Nic overnight (
4ß2 AChRs expressed in HEK cells (
3ß2 or
3
5ß2 AChRs required exposure to Nic before recording, we were unable to determine if the agonist exposure itself altered functionality when compared with naive AChRs. However, the temperature-induced increase in AChRs allowed us to characterize
3ß2 AChRs without prior exposure to Nic (Fig 5 A). The EC50 value for cells incubated at 29°C was 202 ± 14 µM, which compares well to the EC50 of 209 ± 26 µM for cells incubated in Nic (Fig 5 B). Since the functional properties were the same regardless of treatment, it seems unlikely that Nic-treated
3ß2 AChRs have properties that differ from
3ß2 AChRs that have not been Nic-treated. Previous studies with
3ß2 AChRs expressed in Xenopus oocytes showed that this Nic treatment did not alter the functional properties of the AChR (
3ß2 AChRs in the transfected cell line that was reflected by the appearance of substantial electrophysiological responses, overnight incubation of IMR-32 cells in Nic did not increase the amount of
3ß2 AChRs on the surface of these cells (see last paragraph of Subunit Content of AChRs...) or alter their functional properties in a way that might reflect an increase in surface
3ß2 AChRs (Fig 5 C, compare with Fig 1 A, inset). Similar results were found for IMR-32 cells incubated at 29°C (unpublished data).
|
Transfection of 3ß4 and
3ß2 Cell Lines with the
5 Subunit Had Little Effect on the Functional or Pharmacological Properties when Compared with the Parent Cell Line
3
5ß4 transfected cells exhibited pharmacological properties that paralleled closely those of the parent
3ß4 cell line. The agonist rank order of potencies were as follows: DMPP = Cyt > Nic > ACh, and having EC50 values ranging from 20 µM for DMPP to 83 µM for ACh (Fig 4). The agonist efficacies were nearly the same as for the
3ß4 cell line with the exception of Nic (Table 1). Previous immunoprecipitation studies of the
3
5ß4 cell line showed that 14% of the total AChRs expressed the
5 subunit (
3ß4 cell line. Additionally, the partial efficacies of both DMPP and Cyt were nearly identical for
3ß4 and
3
5ß4 cells.
|
The 3
5ß2 cell line had pharmacological properties that differed only slightly from the parent
3ß2 cell line, also. Nic was more potent than ACh on
3
5ß2 cells, but the EC50 for ACh was significantly lower than for the
3ß2 cell line (Fig 4 and Table 1). The rank order of agonist potencies for the
3
5ß2 cell line (DMPP >> Cyt
Nic > ACh) was essentially the same as that for the
3ß2 cell line. Also, the efficacy of Nic was greater for
3
5ß2 AChRs when compared with
3ß2 AChRs. The similar pharmacological properties (Table 1) between these cell lines suggest that the
5 subunit, present in 50% of total AChRs (
5 subunit in
3ß2 AChRs increased both the desensitization rate and the Ca2+ permeability (
Agonist Sensitivity and Efficacy Profiles for 3 Cell Lines and IMR-32 Cells
We used the pharmacological data for AChRs in transfected cells to establish prediction criteria for the presence of either ß2 or ß4 subunits in native human 3 AChRs that are expressed by IMR-32 cells.
3 AChRs that are relatively insensitive to activation by ACh (i.e., have EC50 values greater than 100 µM) and show full efficacy to activation by DMPP are likely to possess ß2 subunits. AChRs where Cyt has significant efficacy and DMPP has less than full efficacy are likely to contain ß4 subunits. The agonist potency profiles found for the
3ß4 and
3
5ß4 cell lines most closely resemble that found for IMR-32 cells (Fig 6 A). Since the
3
5ß4 cell line incorporates
5 subunit, the best match is between IMR-32 and the
3ß4 cell line.
|
The agonist efficacy profiles provide additional support for the conclusion that IMR-32 cells express predominantly 3ß4 AChRs (Fig 6 B). Specifically, Cyt has >70% efficacy on the
3ß4,
3
5ß4, and IMR-32 cells and <10% efficacy on
3ß2 and the
3
5ß2 cell lines, whereas DMPP has >100% efficacy on
3ß2 and
3
5ß2, but <80% efficacy on
3ß4,
3
5ß4, and IMR-32 cells.
Effect of Holding Potential on Agonist Properties for 3ß4 AChRs in HEK Cells
For ACh and Nic, the EC50 values were greater at -30 mV (79 ± 8 µM and 56 ± 10 µM, respectively) than at -60 mV (43 ± 10 µM and 35 ± 6 µM, respectively) whereas for DMPP the EC50 was little changed (15 ± 1 µM at -30 mV and 14 ± 1 µM at -60 mV). Reducing the holding potential also altered the efficacy of Nic and DMPP relative to ACh. The efficacy of Nic peaked between 300 µM and 1 mM at -30 mV, whereas the peak occurred at 100 µM at -60 mV. For DMPP, the efficacy relative to ACh increased from 50 to
65% by depolarizing from -60 to -30 mV. Thus, the effect of holding potential on the concentration-response relationship probably represents the impact of voltage on channel blockade by these agonists, but could also reflect voltage-dependent gating transitions.
All AChRs Exhibited Rectification Regardless of Subunit Composition
Strong inward rectification is a hallmark property of neuronal nicotinic AChRs. It was attributed to intracellular magnesium for -BGTsensitive currents in rat hippocampal neurons (
3ß4 and
4ß2 AChRs (
3 AChR cell lines exhibited inward rectification (Fig 7). The current-voltage relations approach reversal between 0 and +5 mV, but failed to become outward at positive holding potentials up to +60 mV. Recordings at +100 mV with the
3
5ß2 cell line had small outward currents that were
15% of the currents recorded at -100 mV (unpublished data). If polyamines caused the rectification, its persistence throughout 4575-min recordings indicated that the large dilution by electrode solution was insufficient to relieve the channel block. Magnesium or another ion in the electrode solution could also be responsible for the rectification.
|
Desensitization Rate Reflects the ß Subunit Identity in Permanently Transfected HEK Cells
The 3ß2 cells exhibited desensitization that was much faster than that found for
3ß4 cells (Fig 8). Coexpression of the
5 subunit had little effect on the desensitization of the
3ß2 cell line and no effect on the
3ß4 cell line. The
3ß2 currents exhibited both single and double exponential decays. For the
3
5ß2 cell line, desensitization was found to be slightly faster when compared with the
3ß2 cell line. The
3ß4 cell line and the
3
5ß4 cell line had similar decay time constants and both were similar to the value found for IMR-32 AChRs.
|
Single-channel Properties of 3ß4 AChRs Expressed in HEK Cells Closely Match Those of AChRs Expressed by IMR-32
The HEK 3ß4 AChRs had single-channel open times with time constants of 1.9 ± 0.6 and 6.0 ± 1.0 ms (Fig 9). These values were similar to those obtained previously for
3ß4 AChRs recorded from Xenopus oocytes under identical ionic conditions (1.4 ± 0.2 and 6.5 ± 0.8 ms) and similar to the values obtained for IMR-32 AChRs (1.5 ± 0.3 and 9.2 ± 1.2 ms;
3ß4 AChRs were -2.3 ± 0.1 and -1.8 ± 0.1 pA at -80 mV. These amplitudes also were similar to those for both oocyte-expressed
3ß4 AChRs (-2.3 ± 0.1 and -1.6 ± 0.1 pA) and IMR-32 AChRs (-2.2 ± 0.1 pA;
3ß4.
|
Subunit Content of AChRs Expressed by IMR-32 and SH-SY5Y Cells
To estimate the fraction of IMR-32 AChRs that contained a particular subunit, mAb-coated microwells were used. AChRs that contained 3 (and/or
5) subunits (recognized by mAb 210), ß2 subunits (recognized by mAb 295), or ß4 subunits (recognized by mAb 337), were isolated from detergent extracts. mAbs 295 or 337 were tested for their efficiencies in immunoisolating AChRs from the transfected cells. mAb 295 (to ß2) was found to isolate 109 ± 16% as many AChRs as did mAb 210 (to
3) from the
3
5ß2 cell line (Table 2). mAb 337 (to ß4) was found to isolate 64 ± 2% as many AChRs as did mAb 210 (to
3) from the
3ß4 cell line (Table 2). The relatively low efficiency of mAb 337 was confirmed by saturation of immune precipitation using
3ß4 AChRs in extracts from Xenopus oocytes where it bound 61 ± 2% as many AChRs as mAb 210. Increasing mAb 337 concentrations to 100-fold greater than the point of saturation failed to precipitate additional AChRs. This indicated that about one-third of total
3ß4 AChRs existed in which the epitope to mAb 337 on all of the AChR's ß4 subunits is either in the wrong conformation, obscured by another protein, or is consistently proteolyzed.
|
For IMR-32 cell extracts, [3H]epibatidine binding revealed that 65 ± 5% (n = 5) of AChRs bound contained the ß4 subunit when compared with those which contained the 3 subunit, whereas 18 ± 4% contained the ß2 subunit (Fig 10 and Table 2). Additionally, the amount of AChR that could be labeled by [3H]epibatidine on intact IMR-32 cells (4.7 ± 0.8 fmol per 35-mm dish) was not significantly different from the amount of AChR that could immunoisolated from detergent extracts from IMR-32 cells (4.0 ± 0.1 fmol per 35-mm dish). Previously, we found that prolonged incubation of another neuroblastoma cell, SH-SY5Y, in Nic increased total
3 AChRs (
3ß2-transfected cells, but had no effect on
3ß4-transfected cells, and that in SH-SY5Y,
3ß2, but not
3ß4 AChRs were upregulated (
3-containing AChRs by
40% (Fig 10 and Table 2), most of which could be attributed to an increase in ß2-containing AChRs. Similar to previous findings in SH-SY5Y cells, Nic incubation increased the amount of
3 AChRs by
80%, most of which could be attributed to an increase in ß2-containing AChRs.
|
The effects of Nic incubation on surface AChRs in intact cells was determined also. Surface 3-containing AChRs were measured using 125I-mAb 210 and surface ß2-containing AChRs were measured using 125I-mAb 295. In aliquots of IMR-32 cells that bound 10 ± 0.4 fmol of 125I-mAb 210 on their surface, negligible (0.1 ± 0.5 fmol) 125I-mAb 295 binding was detected. Incubation in Nic (100 µM overnight) did not change the amount of binding of either mAb (125I-mAb 210, 10 ± 2 fmol; 125I-mAb 295, 0.4 ± 0.5 fmol). For SH-SY5Y cells, Nic incubation caused no change in the amount of 125I-mAb 210 (4.5 ± 1.1 fmol in control and 4.5 ± 0.4 fmol after Nic incubation) or 125I-mAb 295 binding to the surface of intact SH-SY5Y cells (2.8 ± 0.9 fmol in control and 2.7 ± 0.8 fmol after Nic incubation). Nic incubation had no effect on the properties of currents recorded from IMR-32 cells (Fig 5 C), which is consistent with the surface binding results. Thus, the increase in
3ß2 AChRs found in detergent extracts must reflect an increase in intracellular AChRs.
Immunofluorescence and Confocal Microscope
Confocal microscopy was used to visualize the distribution of 3 AChRs in IMR-32 neuroblastoma cells and the
3ß2 or the
3ß4 transfected cells. For the transfected cells, intense, clustered labeling was observed with antibody directed against the
3 subunit. The labeling of
3 largely overlapped with the labeling observed with antibody directed against the ß4 subunit in the
3ß4 cells and the ß2 subunit in the
3ß2 cells (Fig 11). In the case of IMR-32 cells, the labeling of
3 subunit was also extensive and appeared in clusters that overlapped with the labeling observed for antibody directed at the ß4 subunit (Fig 11). Since mAb 337 (to ß4) targets a cytoplasmic epitope, all images are shown for permeabilized cells. The clustered appearance of the
3 label was also observed on nonpermeabilized cells, indicating that surface AChRs were expressed in concentrated densities (unpublished data). The
3ß4 cells seem to have the largest size clusters, which probably reflects the fact that these cells express the greatest levels of AChR (
|
Western Blot Analysis of Immunoisolated AChRs
AChRs from IMR-32 cells, SH-SY5Y cells, the 3
5ß2 cell line, and the
3ß4 cell line were subjected to immunoisolation followed by Western blotting to establish subunit associations.
3 or ß2 proteins were bound with subunit specific rat antisera raised against the
3 subunit or the ß2 subunit and then labeled with 125I-GART. ß4 protein was labeled directly with 125I-mAb 337. AChRs isolated by mAb 210 from IMR-32 cells or from the
3
5ß2 transfected cell line gave strong bands when labeled with
3 subunit antiserum (Fig 12). AChRs isolated by mAb 295 from the
3
5ß2 cell line revealed strong labeling on Western blots with
3 or ß2 (unpublished data) subunit antisera. However, with IMR-32 cells, relatively weak labeling by
3 or ß2 (not shown) subunit antisera was observed for AChRs isolated by mAb 295 when compared with
3 subunit isolated by mAb 210, which indicated low levels of
3 subunit associated with ß2 subunit. AChRs isolated by mAb 337 resin exhibited strong labeling from both the
3ß4 cell line and from IMR-32 cells relative to
3 subunit isolated by mAb 210, which was consistent with most
3 subunits forming AChRs with ß4 subunits (Fig 12). 125I-mAb 337 labeling of AChR isolated with mAb 210 resin gave stronger signals than for AChR isolated from the same extract by mAb 337 resin, which reflects the difference in mAb efficiencies. These results confirm that IMR-32 cells express AChRs consisting largely of
3 and ß4 subunits and that only very low levels of the ß2 subunit are associated with the
3 subunit. Previous attempts to immune isolate AChRs from IMR-32 extracts with antisera to
4 or
6 subunits or mAb 299 (for
4) failed, reflecting their absence (
|
Presence of a-BGT Binding AChRs in IMR-32 Cells That Do Not Contain 3,
5, or ß2 Subunits
IMR-32 cells have 2.6 ± 0.6 (n = 4) times more total -BGT binding sites than epibatidine binding sites as measured by RIAs of detergent extracts. In a typical experiment, 34 ± 0.6 fmol of 125I-
BGT binding sites of
7-containing AChRs were immunoisolated on microwells coated with mAbs 306 and mAb 319, whereas 14 ± 0.6 fmol of [3H]epibatidine binding sites of
3-containing AChRs were immunoisolated on mAb 210-coated microwells from equal volumes of the same extract. Furthermore, on the cell surface, there are 1.7 ± 0.2 (n = 3) times more binding sites for 125I-
-BGT when compared with 125I-mAb 210. Thus, it was unclear why the functional properties failed to reflect the presence of
7 containing AChRs. However, if the ratio of
-BGT binding sites per AChR is 5:1 and the ratio of mAb 210 binding sites per AChR is 2:1, then there would be similar amounts of
7 and
3 AChRs on the surface. For an AChR with the rapid kinetics of a homomeric
7 AChR, this amount of AChR might not generate sufficient current for detection (see DISCUSSION).
It is possible that IMR-32 cells express heteromeric 7-containing AChRs with slower kinetics that are insensitive to inhibition by MLA. To detect such an AChR we used microwells coated with either mAb 210 (to
3 or
5 subunits) or mAb 295 (to ß2 subunits) and tested for
-BGT binding. For AChRs isolated with either mAb, no 125I-
-BGT binding was detected (Fig 13), but wells coated with
7-selective mAbs 306 and 319 bound 40 ± 4 fmol of 125I-
-BGT from these same extracts. We conclude that in IMR-32 cells, no
-BGT binding AChRs are formed by the coassembly of
3,
5, or ß2 subunits with the
7 AChR subunit.
|
We also tested the effect of overnight Nic (100 µM) treatment and nerve growth factor treatment (0.1 µg/ml for 2 d) on the amount of 7 AChR expressed by IMR-32 cells. Nic treatment reduced slightly the total amount of
7 AChR in IMR-32 cells, whereas NGF had no effect (Fig 13).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IMR-32 cells were derived from an abdominal tumor (3,
5,
7, ß2, and ß4 (
3ß4 AChRs expressed in transfected HEK cells. No evidence was found for
3ß2 or
7 AChR-mediated currents from IMR-32 cells.
3ß2 AChRs represented a small percentage of total IMR-32
3-AChRs and were absent from the cell surface. However, the amount of surface
7 AChRs appeared to be similar to the amount of surface
3 AChRs. The absence of function for
3ß2 or
7 AChRs could result from differential downregulation of these AChRs in the present developmental state of these cells. Incubation in Nic or culturing at reduced temperature increased the amount of
3ß2 AChRs in IMR-32 cells, but not on their surface, in spite of the dramatic increase in surface expression observed for
3ß2 AChRs expressed in HEK cells after these treatments.
Transgenic deletions in mice of the 3, ß2, and ß4 subunits provided evidence that each of the subunits can contribute to AChRs that mediate autonomic transmission (
3 AChR subtypes might play. Also, the morbidity induced by
3 deletion shows the inability of
7 AChRs alone to sustain adequate autonomic synaptic function and provides indirect evidence for distinct roles played by these AChRs. A better understanding of functional differences among AChR subtypes is key to clarifying their roles played in the physiology of the autonomic nervous system.
Comparisons between the Functional and Pharmacological Properties of 3 AChRs Expressed by Permanently Transfected HEK Cells and IMR-32 Cells
The rank order of agonist potencies on IMR-32 cells (DMPP > Nic Cyt = ACh) was similar to reports for ganglionic preparations from both rat and chicken (
7-selective concentrations of methyllycaconitine, the potent reversible competitive antagonist of
7-containing AChRs.
In transfected cells, the rank order of potencies for 3ß4 AChRs was DMPP = Cyt > Nic > ACh, which was similar to the rank order for IMR-32 AChRs. Moreover, with the exception of Cyt, the EC50 values found for agonists on
3ß4 AChRs were nearly identical to those found for IMR-32 AChRs (Table 1). Although the rank order of potencies for
3
5ß4 cells was also similar to IMR-32 cells, both of these cell lines express few
5-containing AChRs (
70% efficacy on both
3ß4 cells and IMR-32 cells, but 5% or less on
3ß2 or
3
5ß2 transfected cells. Desensitization kinetics were similar between
3ß4 AChRs and IMR-32 AChRs, both of which were much slower than the decay kinetics of
3ß2 AChRs. Finally, the single-channel open times and channel amplitudes for the HEK-expressed
3ß4 AChRs were nearly identical to those recorded from oocytes and very similar to those recorded from IMR-32 cells (
These functional data failed to eliminate the possibility that IMR-32 AChRs consist of 3 and ß4 subunits coassembled with ß2 and/or
5 subunits since all possible subunit combinations were not tested in permanently transfected cell lines. It is conceivable, for example, that coassembly with ß4 might mask the presence of ß2 subunits in IMR-32 AChRs. However, the present and past molecular evidence help to dispel this concern. Here, we have shown that mAb 295 (specific for the ß2 subunit) isolated <20% as many AChRs as mAb 210 (specific for
3 AChRs) from IMR-32 cell detergent extracts and it labeled no AChRs on the cell surface (see last paragraph of Subunit Content of AChRs... and next section). Previously, we showed that the amount of
5-containing AChRs in IMR-32 cells is 5% when compared with the amount of
3-containing AChRs (
3 and ß4 subunits. Furthermore, the similarities in functional properties between IMR-32 AChRs and
3ß4 AChRs expressed in HEK cells illustrate two important points: (1) the population of functional IMR-32 AChRs is homogenous, and (2) the expression environment does not appear to modify the functional properties of
3ß4 AChRs.
Effects of Chronic Nic Exposure on AChRs of IMR-32 and SH-SY5Y
Previous studies demonstrated that Nic (micromolar range) incubation increased total 3 AChRs while the number of surface AChRs was unchanged in SH-SY5Y cells (
3ß2 AChRs (
3ß2 AChRs since no effect was seen on human
3ß4 AChRs (
3ß2 AChRs are substantially greater than the concentrations that are typically achieved in serum of cigarette smokers (
3ß2 AChRs by high concentrations of Nic reveals a mode of modulation that could be common among AChRs that contain ß2 subunits. The upregulation has been attributed to promoting AChR assembly coupled with reduced AChR turnover (
3ß2 AChRs in HEK cells, but they caused low levels of upregulation in the absence of Nic (
3-containing AChRs in IMR-32 cells to a lesser extent than those in SY-SY5Y cells. The difference in the extent of upregulation is consistent with the relative abundance of the ß2 subunit in each of the neuroblastoma cells. In fact, ß2-containing AChRs accounted for the majority of the AChR increase caused by Nic, whereas ß4-containing AChRs were unaffected in both IMR-32 and SH-SY5Y cells.
No significant surface ß2 AChRs were detected by antibody in IMR-32 cells, with, or without, Nic treatment. Also, Nic caused no change in the amount of antibody labeling of surface 3 AChRs. For SH-SY5Y, incubation in Nic caused no significant change in the number of
3- or ß2-containing surface AChRs. Because the total number of AChRs expressed by these cells increased, whereas the number of surface AChRs was unchanged after incubation in Nic, AChRs must accumulate intracellularly as suggested previously (
Why might 3ß2 and
3ß4 AChRs within ganglionic neurons have distinct posttranslational regulatory mechanisms? IMR-32 and SH-SY5Y cells represent noninnervated human ganglionic neurons in a suspended state of maturity. The fact that they express different proportions of
3ß2 and
3ß4 AChRs and that
3ß2 levels can be selectively increased by exposure to Nic or by reducing culture temperature shows that mechanisms exist that specifically regulate expression of
3ß2 AChRs in ganglia at certain times of development, stress, or activity. It is interesting that our
3ß2 cell line increases surface AChRs by these treatments, whereas the neuroblastoma cell lines do not. This could mean that other factors such as chaperone proteins or cytoskeletal-associated proteins (analogous to rapsyn for muscle AChRs [
3ß2 AChR expression could be used to achieve these different goals. Comparisons between
3ß2 and
3ß4 cell lines reveal clear functional differences between these AChRs.
3ß2 AChRs are less sensitive to activation and desensitize much more quickly than
3ß4 AChRs. Additionally,
3 AChRs containing the ß2 subunit have higher Ca2+ permeability than
3 AChRs containing the ß4 subunit (
and
subunits in fetal and adult muscle AChR, respectively. AChRs having the
subunit exhibit smaller conductance, longer gating periods, and are not confined to the neuromuscular junction when compared with
-containing AChRs (
3ß2 and
3ß4 AChRs are not significantly different, the gating kinetics differ significantly with the ß4-containing AChRs exhibiting much longer open and burst times than
3ß2 AChRs (
3 AChR subtypes to play specialized roles in ganglionic neurons.
Both neuroblastoma cell lines produce low amounts of 5-containing AChRs. For IMR-32 cells,
5% of
3 AChRs contained the
5 subunit. Such low levels would be insufficient to alter macroscopic currents. However, the same reasoning that was applied to regulated expression of ß2-containing AChRs in ganglia could also be applied to
5-containing AChRs. Considering the impact of the
5 subunit on
3-AChR gating, conductance, and permeability (
Expression of 7 AChRs in IMR-32 Cells with No Detectable Function
-BGTsensitive currents have been reported in IMR-32 cells previously (
-BGT blockade, these data are difficult to interpret. In the present study, no IMR-32 currents had rapid kinetics like those of an
7-type response (
7 AChRs to validate the efficiency of our application system (unpublished data). Additionally, we failed to inhibit Nic-evoked currents in IMR-32 cells with the reversible selective
7 AChR antagonist, MLA. Thus, the presence of
-BGT binding sites measured on the cell surface in IMR-32 cells was perplexing. However, if
7 AChRs have an
-BGT binding site for each subunit of a homopentameric AChR, there would be similar numbers of
7 AChRs when compared with
3 AChRs on the cell surface. Considering that the channel closing rate for homomeric
7 AChRs is at least 10,000 s-1 ([
150 s-1 for
3ß4 AChRs [ Fig 9]) and its desensitization rate is at least 3000 s-1 (unpublished data; compared with
2 s-1 for
3ß4 AChRs [ Fig 8]) this number of
7 AChRs might fail to generate measurable current. Consistent with this reasoning, we estimate that there are sevenfold fewer
-BGT binding sites in IMR-32 cells than have been reported for transfected HEK cells from which human
7 AChRs have been characterized electrophysiologically (
7 AChRs expressed by IMR-32 cells are, indeed, nonfunctional. To test for the presence of nonhomomeric
7 AChRs with unexpected functional properties, we immunoisolated AChRs that contained either
3,
5, or ß2 subunits, and then attempted to label them with 125I-
-BGT. No labeling was observed on these AChRs, but
7-selective mAbs 306 and 319 bound 40 ± 4 fmol of 125I-
-BGT from these same extracts confirming the presence of an
7 AChR with high affinity for toxin.
Conclusion
The physiological significance of the potential for such functional diversity among AChRs within individual neurons remains unclear. 7 AChRs are highly permeable to Ca2+, which makes them good candidates for triggering Ca2+-dependent events to alter expression of proteins involved in morphological or functional changes. The same may also be true of
3ß2 AChRs as it has been shown previously that
3ß2 AChRs have higher Ca2+ permeability than
3ß4 AChRs. In fact, the more Ca2+-permeable AChRs might mediate development and plasticity, whereas
3ß4 AChRs mediate excitatory synaptic transmission. In any case, it is possible that relative amounts of AChR subtypes expressed might vary in neurons from one ganglion to the next. Our data indicate that IMR-32 cells express
3ß4 AChRs as the predominant functional AChRs. What remains to be elucidated is what mechanisms are used by ganglionic neurons to alter the proportions of AChR subtypes in response to changing cellular needs. Both incubation in Nic and temperature reduction provide useful models through which to study at least part of these regulatory mechanisms.
![]() |
Footnotes |
---|
The current address of F. Wang is DuPont Central Research and Development, Experimental Station, Wilmington, DE 19880. The current address of V. Gerzanich is Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, MD 21201.
* Abbreviations used in this paper: AChR, acetylcholine receptor; Cyt, cytisine; HEK, human embryonic kidney; MLA, methyllycaconitine; Nic, nicotine; RIA, radioimmune assay.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The authors thank John Cooper and Ben McNeil for their technical assistance.
This work supported by grants to J. Lindstrom from the National Institutes of Health (NS11323) and the Smokeless Tobacco Research Council, Inc., USA, Inc.
Submitted: 30 April 2001
Revised: 28 September 2001
Accepted: 1 October 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alkondon, M., Pereira, E.F., Wonnacott, S., and Albuquerque, E.X. 1992. Blockade of nicotinic currents in hippocampal neurons defines methyllycaconitine as a potent and specific receptor antagonist. Mol. Pharmacol. 41:802-808[Abstract].
Anand, R., Nelson, M.E., Gerzanich, V., Wells, G.B., and Lindstrom, J. 1998. Determinants of channel gating located in the N-terminal extracellular domain of nicotinic 7 receptor. J. Pharmacol. Exp. Therap. 287:469-479[Abstract/Full Text].
Benowitz, N., Porchet, H., and Jacob, P. 1990. Pharmocokinetics, metabolism, and pharmacodynamics of nicotine. In Wonnacott S., Russell M., Stolerman I., eds. Nicotine Psychophamacology. Oxford, England, Oxford Science Publications, 112-157.
Betz, H., Kuhse, J., Schmieden, V., Malosio, M.L., Langosch, D., Prior, P., Schmitt, B., and Kirsch, J. 1991. How to build a glycinergic postsynaptic membrane. J. Cell Sci. Suppl 15:23-25[Medline].
Bloch, R.J., and Froehner, S.C. 1987. The relationship of the postsynaptic 43K protein to acetylcholine receptors in receptor clusters isolated from cultured rat myotubes. J. Cell Biol. 104:645-654[Abstract].
Cooper, S.T., Harkness, P.C., Baker, E.R., and Millar, N.S. 1999. Up-regulation of cell-surface 4ß2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits. J. Biol.Chem 274:27145-271452[Abstract/Full Text].
Covernton, P.J., Kojima, H., Sivilotti, L.G., Gibb, A.J., and Colquhoun, D. 1994. Comparison of neuronal nicotinic receptors in rat sympathetic neurones with subunit pairs expressed in Xenopus oocytes. J. Physiol. 481:27-34[Abstract].
Franceschini, D., Orr-Urtreger, A., Yu, W., Mackey, L.Y., Bond, R.A., Armstrong, D., Patrick, J.W., Beaudet, A.L., and De Biasi, M. 2000. Altered baroreflex responses in 7 deficient mice. Behav Brain Res 113:3-10[Medline].
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. Therap. 286:311-320[Abstract/Full Text].
Gopalakrishnan, M., Buisson, B., Touma, E., Giordano, T., Campbell, J.E., Hu, I.C., Donnelly-Roberts, D., Arneric, S.P., Bertrand, D., and Sullivan, J.P. 1995. Stable expression and pharmacological properties of the human 7 nicotinic acetylcholine receptor. Eur. J. Pharmacol. 290:237-246[Medline].
Gotti, C., Briscini, L., Verderio, C., Oortgiesen, M., Balestra, B., and Clementi, F. 1995. Native nicotinic acetylcholine receptors in human imr32 neuroblastoma cells: functional, immunological and pharmacological properties. Eur. J. Neurosci. 7:2083-2092[Medline].
Gotti, C., Fornasari, D., and Clementi, F. 1997. Human neuronal nicotinc receptors. Prog. Neurobiol. 53:199-237[Medline].
Haghighi, A.P., and Cooper, E. 1998. Neuronal nicotinic acetylcholine receptors are blocked by intracellular spermine in a voltage-dependent manner. J. Neurosci. 18:4050-4062[Abstract/Full Text].
Haghighi, A.P., and Cooper, E. 2000. A molecular link between inward rectification and calcium permeability of neuronal nicotinic acetylcholine 3ß4 and
4ß2 receptors. J. Neurosci. 20:529-541[Abstract/Full Text].
Hall, Z.W., and Sanes, J.R. 1993. Synaptic structure and development: the neuromuscular junction. Cell. 72(Suppl):99-121[Medline].
Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391:85-100[Medline].
Kuryatov, A., Olale, F.A., Choi, C., and Lindstrom, J. 2000. Acetylcholine receptor extracellular domain determines sensitivity to nicotine-induced inactivation. Eur. J. Pharmacol. 393:11-21[Medline].
Lane, R.D., Crissman, R.S., and Ginn, S. 1986. High efficiency fusion procedure for producing monoclonal antibodies against weak immunogens. Methods Enzymol. 121:183-192[Medline].
Mandelzys, A., Pie, B., Deneris, E.S., and Cooper, E. 1994. The developmental increase in ACh current densities on rat sympathetic neurons correlates with changes in nicotinic ACh receptor -subunit gene expression and occurs independent of innervation. J. Neurosci. 14:2357-2364[Abstract].
McGehee, D.S., and Role, L.W. 1995. Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Phys. 57:521-546.
Nelson, M.E., and Lindstrom, J. 1999. Single channel properties of human 3 AChRs: impact of ß2, ß4 and
5 subunits. J. Physiol. 516:657-678[Abstract/Full Text].
Orr-Urtreger, A., Goldner, F.M., Saeki, M., Lorenzo, I., Goldberg, L., De Biasi, M., Dani, J.A., Patrick, J.W., and Beaudet, A.L. 1997. Mice deficient in the 7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J. Neurosci. 17:9165-9171[Abstract/Full Text].
Palma, E., Bertrand, S., Binzoni, T., and Bertrand, D. 1996. Neuronal nicotinic 7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine. J. Physiol. 491:151-161[Abstract].
Papke, R.L., and Heinemann, S.F. 1994. Partial agonist properties of cytisine on neuronal nicotinic receptors containing the ß2 subunit. Mol. Pharmacol. 45:142-149[Abstract].
Peng, X., Katz, M., Gerzanich, V., Anand, R., and Lindstrom, J. 1994. Human 7 acetylcholine receptor: cloning of the
7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional
7 homomers expressed in Xenopus oocytes. Mol. Pharmacol. 45:546-554[Abstract].
Peng, X., Gerzanich, V., Anand, R., Wang, F., and Lindstrom, J. 1997. Chronic nicotine treatment up-regulates 3 and
7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y. Mol. Pharmacol. 51:776-784[Abstract/Full Text].
Ross, R.A., Spengler, B.A., and Biedler, J.L. 1983. Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J. Natl. Cancer Inst. 71:741-747[Medline].
Rust, G., Burgunder, J.M., Lauterburg, T.E., and Cachelin, A.B. 1994. Expression of neuronal nicotinic acetylcholine receptor subunit genes in the rat autonomic nervous system. Eur J. Neurosci. 6:478-485[Medline].
Sargent, P.B. 1993. The diversity of neuronal nicotinic acetylcholine receptors. Ann. Rev. Neurosci. 16:403-443[Medline].
Tumilowicz, J.J., Nichols, W.W., Cholon, J.J., and Greene, A.E. 1970. Definition of a continuous human cell line derived from neuroblastoma. Cancer Res. 30:2110-2118[Medline].
Ullian, E.M., McIntosh, J.M., and Sargent, P.B. 1997. Rapid synaptic transmission in the avian ciliary ganglion is mediated by two distinct classes of nicotinic receptors. J. Neurosci. 17:7210-7219[Abstract/Full Text].
Wang, F., Gerzanich, V., Wells, G.B., Anand, R., Peng, X., Keyser, K., and Lindstom, J. 1996. Assembly of the human neuronal nicotinic receptor 3 subunit with ß2, ß4, and
5 subunits. J. Biol. Chem. 271:17656-17665[Abstract/Full Text].
Wang, F., Nelson, M.E., Kuryatov, A., Olale, F., Cooper, J., Keyser, K., and Lindstrom, J. 1998. Chronic nicotine treatment upregulates human 3ß2, but not
3ß4 AChRs stably transfected in human embryonic kidney cells. J. Biol. Chem. 273:28721-28732[Abstract/Full Text].
Wang, H., Bedford, F.K., Brandon, N.J., Moss, S.J., and Olsen, R.W. 1999. GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton. Nature. 397:69-72[Medline].
Whiting, P.J., and Lindstrom, J.M. 1988. Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies. J. Neurosci. 8:3395-3404[Abstract].
Xu, W., Gelber, S., Orr-Urtreger, A., Armstrong, D., Lewis, R.A., Ou, C.N., Patrick, J., Role, L., De Biasi, M., and Beaudet, A.L. 1999a. Megacystis, mydriasis, and ion channel defect in mice lacking the 3 neuronal nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA. 96:5746-5751[Abstract/Full Text].
Xu, W., Orr-Urtreger, A., Nigro, F., Gelber, S., Sutcliffe, C.B., Armstrong, D., Patrick, J.W., Role, L.W., Beaudet, A.L., and De Biasi, M. 1999b. Multiorgan autonomic dysfunction in mice lacking the ß2 and the ß4 subunits of neuronal nicotinic acetylcholine receptors. J. Neurosci. 19:9298-9305[Medline].