The Chaperone Protein 14-3-3η Interacts with the Nicotinic Acetylcholine Receptor α4 Subunit

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) α4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3η, known to interact directly with neuronal AChRs. 14-3-3η is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3η interacted with the recombinant α4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3η with recombinant α4 subunits was abolished when serine 441 of the α4 subunit was mutated to alanine (α4S441A). The surface levels of recombinant wild-type α4β2 AChRs were ∼2-fold higher than those of mutant α4S441Aβ2 AChRs. The interaction significantly increased the steady state levels of the α4 subunit and α4β2 AChRs but not that of the mutant α4S441A subunit or mutant α4S441Aβ2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for α4β2 AChRs and α4S441Aβ2 AChRs coexpressed with 14-3-3η in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native α4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of α4β2 AChRs through its interaction with the α4 subunit.


INTRODUCTION
Neuronal nicotinic acetylcholine receptors (AChR) 1 are a family of ligand-gated, cation-selective, homo-or heteropentameric ion channels expressed in the peripheral and central nervous system (1,2). A multitude of neuronal AChR subtypes assembled from different combinations of α2-α9 and β2-β4 subunits have been identified (3,4). Of these, the α4β2 AChR is widely expressed in the CNS and represents >80% of the high-affinity [ 3 H] nicotine-binding sites in mammalian brain (5). Our understanding of their physiological roles comes most recently from gene knock-out studies in mice. Mice in which the α4 subunit gene has been deleted lack [ 3 H] nicotine or [ 3 H] epibatidine binding sites in their brain and exhibit reduced antinociceptive effects of nicotine (6). Mice in which the β2 subunit gene has been deleted also show little [ 3 H] nicotine binding in their brains, lose their sensitivity to nicotine in passive avoidance tasks (7), and show attenuated self-administration of nicotine (8) suggesting that α4β2 AChRs have a role in mediating addiction to nicotine. The normal and pathophysiological functions mediated by α4β2 AChRs are of significant importance to human health. Some inherited forms of epilepsy, such as the autosomal dominant nocturnal frontal lobe epilepsies, are caused by α4β2 AChRs harboring at least two separate mutations within their α4 subunit (9)(10)(11)(12).
Most recently, α4β2 AChRs, among other β2 subunit-containing AChRs, have been 14-3-3η Stabilizes Nicotinic Receptor α4 Subunits 9 Bethesda, MD) at 90% confluency (~10 6 cells/well) with various combinations of cDNAs as per the manufacturer's instructions and utilized after ~48h. The cDNAs were cloned into the vector pEF6/myc-His but lacked the myc-His tag because of the presence of the endogeneous stop codon present in each cloned cDNA. AChR subunit assembly was found to be more efficient at 30 o C than at 37 o C as previously described (17) and hence the experiments were performed with cells incubated at 30 o C following transfection.

Expression and analysis of the 4 subunit.
To study the effect of 14-3-3η on the α4 subunit alone, tsA201 cells seeded (400,000 per well) in 12 wells, were incubated at 37˚C. The next day the cells were cotransfected with α4 or α4 S441A with or without 14-3-3η and incubated at 30˚C (the DNA concentrations and ratios were kept constant by using the pEF6A vector DNA). The transfected cells were treated 24 h with or without forskolin (10µM). After washing once with ice cold PBS, the cells were solubilized in 500µl of the following lysis buffer: 50mM NaCl, 30mM triethanolamine, pH 7.5, 5mM EGTA, 5mM EDTA, 1mM benzamidine, 5µg/ml aprotinin, 5µg/ml leupeptin, 5µg/ml pepstatin and 2% NP-40. After shaking for 3 hours at 4˚C, the lysates were centrifugated for 15 min. at 18,000xg and 20µl of the supernatant were analyzed by SDS-PAGE.
Immunoisolation of native AChRs from rat brain. Frozen rat brains were homogenized in 10 vol of homogenization buffer (50mM NaCl, 30mM triethanolamine, pH 7.5, 5mM EGTA, 5mM EDTA, 50mM NaF, 1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, 2mM sodium vanadate, 10mM p-nitrophenylphosphate) using a homogenizer (OMNI International, Warrenton, VA). The homogenate was centrifuged at 100, 000 xg in a Beckmann 50.2 Ti rotor for 30 min at 4 o C. The membrane pellet was further briefly homogenized and then extracted with 3 volumes of a solubilization buffer (homogenization buffer containing 1% NP-40 and 25µg/ml aprotinin, 25µg/ml leupeptin, 25µg/ml pepstatin, 0.1µM okadaic acid, 1mM sodium tetrathionate, 1mM Nethylmaleimide, 50µM phenylarsine oxide) for 2 h at 4 o C. The clear supernatant obtained after centrifugation of the pellet at 18, 000xg for 30 min was used for all subsequent immunoisolation procedures. Detergent-solubilized brain extracts (typically 10 ml) thus obtained were incubated with approximately 25µl of mAb-coupled Actigel ALD bead by guest on March 22, 2020 http://www.jbc.org/ Downloaded from 14-3-3η Stabilizes Nicotinic Receptor α4 Subunits 11 (that were preblocked with 5% non-fat milk for 30 min) at 4 o C for 72 h. In initial experiments, to ensure that the binding observed was specific, we first determined the number of successive washes of the mAb-beads necessary for the complete removal of unbound 14-3-3 proteins which are abundant in brain extracts and many of whose isoforms crossreact with the anti-14-3-3 mAb used in the immunoblotting experiments.
The beads thus were typically washed 10 times with ~800 µl of solubilization buffer and eluted with sample buffer (lacking β-mercaptoethanol to avoid reduction of the disulphide linkage of the IgG chains) at 60 o C for 30 min and then β-mercaptoethanol was added to the eluted samples prior to analysis by SDS-PAGE.
Immunoblot analysis. The proteins bound to the Ab-beads were eluted with protein sample buffer and fractionated by SDS-PAGE. The proteins were electroblotted onto polyvinylidene difluoride membrane (IMMUN-BLOT; Bio-Rad Laboratories, Hercules, CA) and the membranes incubated with diluted (typically 1:200 to 1:1000) primary Abs in phosphate-buffered saline solution containing 0.1% Tween and 5% non-fat milk. The binding of the primary mAbs was detected using appropriate secondary Abs conjugated to horseradish peroxidase in conjunction with a chemiluminescence detection kit (SuperSignal, Pierce). To reduce nonspecific binding, the blots were typically cut in half and the top half probed with the anti-α4 subunit mAb and the bottom half with the anti-14-3-3 mAb thus eliminating the need for sequential reprobing of the blots.

Enzyme-linked immunoassay for cell surface AChRs.
Cell surface α4β2 AChRs were measured as previously described (17). Briefly, 48 h after transfection, tsA201 cells by guest on March 22, 2020 http://www.jbc.org/ Downloaded from 14-3-3η Stabilizes Nicotinic Receptor α4 Subunits 12 plated in 12 well plates (0.5x10 6 cells/well) were washed once in PBS, then blocked with PBS containing 3%BSA and the cells were incubated for 1h with an anti-β2 subunit mAb (295) in PBS containing 3%BSA at room temperature. After four washes with PBS the cells were fixed with formaldehyde (3%) for 10 min, washed three times with PBS and blocked again for 10 min. The cells were then incubated with horseradish peroxidaseconjugated goat anti-rat secondary Ab for 1 h in the presence of 3% BSA, washed six times with PBS, and incubated with 300µl of the HRP substrate 3, 3', 5, 5'tetramethylbenzidine (Sigma) for 1 h. The absorbance of the supernatant was then measured at 655nm in a Beckmann spectrophotometer.
Expression in Xenopus oocytes. cDNAs were subcloned into the vector pSP64T (Invitrogen) with a modified polylinker. cRNAs from linearized cDNA templates were synthesized in vitro using SP6 RNA polymerase in conjunction with reagents from the mMessage mMachine kit (Ambion, Austin, TX). Xenopus oocytes were prepared for injection as previously described (18). Oocytes were injected with 20ng of cRNAs for the α4 and β2 subunits and 40ng of 14-3-3η per oocyte and incubated for 3-7 days at 16-18 o C in 50% L-15 medium (Life Technologies Inc.) containing 10mM HEPES buffer, pH 7.5.

14-3-3 interacts with the large cytoplasmic domain of the 4 subunit in the yeast twohybrid system
The large cytoplasmic domain corresponding to amino acids 302-561 of the rat AChR α4 subunit was used as a bait to screen ~2x10 6 clones of a mouse brain cDNA LexA yeast two-hybrid library. The large cytoplasmic domain extends from the third to the fourth transmembrane domain of the AChR α4 subunit. Multiple clones of 14-3-3η that interacted with the α4 bait were obtained. A full-length clone of 14-3-3η was chosen for further characterization.
To delineate the site at which 14-3-3η interacts with the AChR α4 subunit cytoplasmic domain, a series of C-terminal nested deletions of the cytoplasmic loop was created as LexA fusion protein baits and tested for their ability to interact with the 14-3-3η clone in the yeast two-hybrid system. The interaction was determined by both the ability of transformed yeast cells to grow on media lacking leucine, tryptophan, histidine, and uracil and by their ability to turn blue on media supplemented with X-gal. As controls, the LexA protein alone was used as a bait. We mapped the interaction of 14-3-3η to residues 413 to 450 ( Figure 1A). A putative motif, RSXSXP, in which the underlined serine residue is phosphorylated, has been previously demonstrated to be important for the binding of 14-3-3 proteins to some of its target proteins. A sequence that closely resembles this motif, RSLSVQ, occurs within the region 413-450 that was within a specific consensus binding site motif.

14-3-3 interacts with recombinant 4 subunits in tsA 201 cells
To directly test if 14-3-3η could interact with full-length unassembled α4 subunits alone, we transfected tsA 201 cells with the α4 subunit cDNA. Because it has been previously demonstrated that phosphorylation of a consensus binding site motif for 14-3-3 greatly increases its affinity for the site, we also tested if activation of kinases would increase the interaction of 14-3-3η with the recombinant α4 subunit. We treated transfected cells with the idea that PKA-dependent phosphorylation was involved in mediating the interaction.

14-3-3 interacts with recombinant 4 2 AChRs expressed in tsA 201 cells
To determine whether 14-3-3η could also interact with recombinant α4β2 AChRs in mammalian cells, we immunoisolated 1%NP-40-solubilized recombinant α4β2 AChRs using anti-α4 subunit mAb beads from tsA 201 cells transfected with the α4, β2, and 14-3-3η cDNAs. As a control for nonspecific binding, we used beads coupled to nonspecific rat IgG. We observed immunoreactivity for 14-3-3η and the α4 subunit migrating at their expected molecular masses of ~30kDa and ~70kDa, respectively ( The preceding results were obtained by immunoisolating recombinant α4β2 AChRs with anti-α4 mAb beads. Since this mAb binds both assembled and unassembled α4 subunits, we were unable to distinguish if 14-3-3η interacted with α4 subunits that were unassembled, or assembled with the β2 subunits. To test if 14-3-3η could interact with assembled α4 subunits, α4β2 complexes were isolated with the anti-β2 mAb beads. The anti-β2 mAb used (mAb 295) binds the conformationally mature β2 subunit only.
Reactivity with denatured β2 subunits on immunoblots is not observed. We found that activation of PKA by forskolin significantly enhanced the interaction of 14-3-3 with α4β2 AChR complexes immunoisolated with the anti-β2 mAb beads both in the absence . We also observed low basal levels of interaction of 14-3-3 with α4β2 AChR complexes even prior to activation of PKA by forskolin (Fig 3B, lanes 1 and 3). These results suggested that both endogeneous 14-3-3 and exogeneous 14-3-3η associated with the α4β2 AChR complexes. In addition, in three independent experiments we also observed that both in the presence or absence of exogeneous 14-3-3η, the amount of α4 subunits immunoprecipitated by the anti-β2 mAb beads from detergent extracts of cells treated with forskolin was consistently higher than that from detergent extracts of cells not treated with forskolin. The increased amounts of the α4 subunits also correlated with greater amounts of 14-3-3 coimmunoprecipitated with the α4β2 AChR complexes using the anti-β2 mAb beads suggesting a possible role for 14-3-3η in altering the steady state levels of α4β2 AChR complexes. To further demonstrate that the interaction of 14-3-3η with the α4β2 AChRs was phosphorylation dependent, the detergent-solubilized α4β2 AChR immunoisolated complexes from cells treated with forskolin and IBMX, were treated with recombinant protein phosphatase I (PPI). We observed a significant reduction in the amount of 14-3-3η associated with immunopurified recombinant α4β2 AChRs treated with PPI compared to those treated with the buffer alone under identical conditions (Fig. 3C). These results further supported the fact that forskolin-dependent enhancement of the interaction of 14-3-3η with α4β2 AChRs was due to a PKA-mediated phosphorylation event.

14-3-3 stabilized the wild-type 4 subunit but not the mutant 4 S441A subunit
Since 14-3-3η bound to the α4 subunit alone, we examined if 14-3-3η, a chaperone protein had a role in the early biogenesis of the α4 subunit. The α4 and the α4 S441A subunits were separately cotransfected with or without 14-3-3η cDNA into tsA201 cells.
We studied the influence of the presence of 14-3-3η on the α4 or α4 S441A subunit steady state levels prior to, and following, activation of PKA by forskolin. To ensure differences were not simply due to variability in transfections between wells treated similarly, each condition was independently processed and analyzed in duplicate (indicated by a bar over the two lanes in Figure 4).

Higher surface expression of wild-type 4 2 AChRs than mutant 4 S441A 2 AChRs
To determine whether the interaction of 14-3-3η with the α4 subunits altered the cell surface expression levels of α4β2 AChRs , we monitored these levels for both wildtype α4β2 AChRs and mutant α4 S441A β2 AChRs using an enzyme-linked immunoassay.
Because 14-3-3 interacts with α4β2 AChRs following activation of PKA, we also examined if activation of PKA by forskolin (10µM) altered cell surface expression levels of wild-type α4β2 AChRs and mutant α4 S441A β2 AChRs. The modified enzyme-linked immunoassay we used has been used previously to measure the surface expression of α4β2 AChRs (17). In our assay we measured the relative amount of the β2 subunit in cells treated under the described conditions with an anti-β2 subunit primary antibody.
The amount of β2-immunoreactivity was then determined using an HRP-conjugated secondary Ab. The amount of secondary Ab bound to the primary mAb was then determined by measuring HRP enzymatic activity of the conjugated enzyme on a substrate (3, 3', 5, 5'-tetramethylbenzidine) whose product is colored blue, and whose concentration can then be determined spectrophotometrically.
As controls for nonspecific binding of the Abs, we used cells transfected with the vector alone. The surface expression of wild-type α4β2 AChRs was found to be ~2-fold higher than the mutant α4 S441A β2 AChRs ( Figure 5). Following treatment with forskolin (10µM), the wild-type α4β2 AChRs showed a small but statistically significant increase (~20%, n=7, AChRs. The 2-fold difference between the surface expression levels of the α4β2 AChRs and the α4 S441A β2 AChRs was observed with two different preparations of cDNAs making it very unlikely that it was due to differences in transfection efficiencies between the α4 subunit cDNA and the α4 S441A subunit cDNA due to differences in the quality of the DNA samples. Similar results in the absence of transfected exogeneous 14-3-3η (data not shown) in keeping with our findings that the endogeneous 14-3-3 associated with α4β2 AChRs under these conditions .
AChR subunits were expressed from in vitro transcribed cRNAs microinjected into oocytes and currents elicited by 4 sec applications of different concentrations of ACh recorded using two-electrode voltage clamp methodology. ACh elicited dose-dependent response from both wild-type α4β2 AChRs and mutant α4 S441A β2 AChRs when expressed alone or when coexpressed with 14-3-3η and treated with forskolin (50µM) for 4 h at room temperature. The whole cell currents are shown in Fig. 6  for AChR activation were not significantly different, these results suggested that 14-3-3η was unlikely to have a role in modulation of the functional properties of α4β2 AChRs.

Immunohistochemical localization of 14-3-3 and the 4 subunit in transfected cells
We compared the distribution of 14-3-3 proteins with that of the α4 subunit at the single cell level in transfected cells treated with and without forskolin (10µM).
Transfected cells were fixed with methanol/acetone and then sequentially immunostained for the α4 subunit followed by staining for 14-3-3 as described in the experimental procedures section. Antibody binding was then visualized by confocal immunofluorescence microscopy using goat anti-mouse Alexa Fluor 546 conjugated Abs and the goat anti-rat Alexa Fluor 488 conjugated Abs. At the single cell level, diffuse immunostaining for the α4 subunit (red, top panel, Fig 7) was observed throughout the ER/Golgi compartments and the surface membrane. In contrast staining for 14-3-3 was very distinctively different and was confined to the cytosolic region (green, top panel,  Fig. 7). Following treatment with forskolin, colocalization within the ER/Golgi compartments was significantly enhanced, but no significant colocalization was evident at the surface membrane (yellow, bottom panel, Fig 7). As controls, we were coexpresssed with 14-3-3η was most likely because 14-3-3η did not colocalize with surface α4β2 AChRs.

Interaction of 14-3-3 with native 4 2 AChRs from rat brain
To validate the physiological importance of the interaction of 14-3-3η with the α4 subunit in yeast, and with recombinant α4β2 AChRs in transfected cells, we determined if 14-3-3 is associated with native α4β2 AChRs immunopurified from rat brain. Rat brain membranes were solubilized using 1% NP-40 and the α4β2 AChRs immunopurified using anti-α4 subunit-specific mAbs and anti-β2 subunit-specific mAbs. Detergent-solubilized brain membrane extracts were also incubated with beads coupled to a control Ab (rat IgG). The interaction of 14-3-3 with α4β2 AChRs was then detected by immunoblotting with an anti-14-3-3 mAb. 14-3-3 was found to be associated with complexes of native

DISCUSSION
The cloning of a multitude of neuronal AChR subunit cDNAs has revealed a great diversity of AChR subtypes whose functions in the nervous system remain enigmatic (19). The large cytoplasmic domain between the third and fourth transmembrane domain is highly divergent among the subunits (20). Some aspects of the roles subserved by the large cytoplasmic domain such as the polarized trafficking of AChR in neurons (21), the clustering of muscle AChRs at synaptic membrane subsites (22)(23)(24) are known.
Identification of proteins that interact with the cytoplasmic domain is likely to provide a better understanding of proteins involved in the subunit assembly, trafficking, clustering, and functions of AChRs. As a first step toward understanding which proteins interact with the widely expressed neuronal α4 AChRs, we used the α4 subunit cytoplasmic domain in a yeast two-hybrid screen. In this paper, we describe the identification of the first protein known to interact with the α4 subunit, 14-3-3η, and the characterization of its interaction with recombinant and native α4β2 AChRs. The results of our study provide novel mechanistic insights into the cellular events that mediate the interaction of 14-3-3η with the AChR α4 subunit following activation of PKA, and the consequences of this interaction on the stability of the subunit.
14-3-3 proteins have previously been shown to bind to the sequence motif We observed that activation of PKA significantly enhanced the interaction of 14-3-3η with unassembled α4 subunits and with assembled α4β2 AChR complexes. We have however failed to detect an increase in association of 14-3-3η with α4β2 AChRs in tsA201 cells following acute or chronic (24h) exposure of AChRs to nicotine (data not shown). These results suggest that other intracellular processes, other than channel activity, possibly governs the interaction of 14-3-3 with the α4 subunit and α4β2 AChRs.
We have provided compelling evidence for a role of 14-3-3η in increasing the stability of the α4 subunit and α4β2 AChR under conditions that also correlate well with those that favor interaction of 14-3-3 with the α4 subunit. When α4 subunits are expressed alone, the wild-type α4 and mutant α4 S441A subunits did not show significant Corresponding differences in the steady state levels of the α4β2 AChR and the α4 S441A β2 AChRs were also observed and strongly suggested that 14-3-3η plays a role in early posttranslational events that govern subunit and α4β2 AChR stability.
The phosphorylation of the α4 subunit at serine 441 by PKA and its subsequent interaction with 14-3-3 alters cell surface α4β2 AChRs by increasing the α4 subunit and α4β2 AChR steady state levels. In keeping with such a role for 14-3-3, we observed a correlation between higher cell surface expression levels of wild-type α4β2 AChRs and its ability to bind 14-3-3η and lower surface expression levels of the mutant α4 S441A β2 AChRs and their inability to bind 14-3-3η. Furthermore we observed a small but significant increase in their cell surface expression levels following treatment with forskolin. In contrast, forskolin did not induce a significant change in the cell surface expression levels of mutant α4 S441A β2 AChRs. Similar results in surface expression levels following treatment with forskolin were observed in the absence of exogeneous 14-3-3η and were most probably due to the observed ability of endogeneous 14-3-3 proteins to interact with α4β2 AChRs .
Previously, it has been reported that activation of PKA by forskolin results in ã 200% increase in cell surface expression of recombinant human α4β2 AChRs expressed in tsA201 cells (35). However, we do not observe such a large increase in surface expression of rat α4β2 AChRs expressed in tsA201 cells. We suggest that this difference perhaps reflects differences in the growth conditions and species-specific differences (human versus rat) that might also affect the intrinsic efficiency of subunit assembly.
The rather small but statistically significant increase (~20%) in surface expression levels of the wild-type α4β2 AChRs following treatment with forskolin is consistent with the idea that when subunit assembly was efficient, PKA-dependent phosphorylation only marginally contributes to further increases in surface expression.
The role of phosphorylation in regulating subunit assembly and cell surface expression is better characterized for muscle-type AChRs (36)(37)(38)(39)(40)(41)(42)(43). In muscle-type AChRs, pulse chase experiments and immunofluorescent microscopy indicate that AChR subunit assembly is complete in the ER following which AChR oligomers move rapidly through the Golgi membrane onto the plasma membrane (37). Interestingly it has been demonstrated that both the γ and δ subunits are phosphorylated in vivo, and the δ subunits is more highly phosphorylated in the unassembled than in the assembled state indicating that phosphorylation precedes assembly and that phosphorylation/dephosphorylation mechanisms control AChR subunit (36). Furthermore, using Torpedo AChR subunits expressed in mouse fibroblasts, it has been previously demonstrated that cAMP-induced increase in expression of cell surface AChRs is due to phosphorylation of the unassembled γ subunit assembly (37). But the underlying mechanism by which this phosphorylation increases the efficiency of subunit assembly and increased surface AChR expression, has not been elucidated.
We have demonstrated that phosphorylation of the unassembled α4 subunit and the subsequent association of 14-3-3 with it increases it steady state levels in nonneuronal cells. This mechanism is consistent with such a proposed role for 14-3-3 in regulating the turnover of the plasma membrane H + -ATPase (44). In addition, both PKA (45) and 14-3-3 isoforms (46) have been previously demonstrated to be appropriately localized to the ER/Golgi compartments to participate in such a process. Our results do not identify which exact isoform(s) of 14-3-3 is associated with the native α4 AChR subunit because the anti-14-3-3 mAb we used cross-reacts with several members of the 14-3-3 family.
The family of 14-3-3 proteins consists of closely related members that do not show measurable differences in their affinities for a consensus binding site motif in vitro (33), though their binding in vivo is regulated by modulating their expression levels (47)(48)(49) and by phosphorylation of the 14-3-3 proteins themselves (50)(51)(52). Thus, the identity of the particular isoform(s) of 14-3-3 that binds the native α4 AChR subunit in vivo, remains to be determined. Because 14-3-3 can dimerize and thus simultaneously bind two different proteins, further experimentation will be needed to establish if other proteins are also involved in this process.
Finally, we would like to point out a possible pathophysiological significance of our work. It is well established that chronic intake of nicotine in smokers increases the expression levels of α4β2 AChRs in their brains (53 Interestingly, recent genetic analyses of allelic frequencies of a variable number of tandem repeat in the 5'-noncoding region of the 14-3-3η gene suggests that it is a potential susceptibility gene for schizophrenia, particularly for early-onset schizophrenia (55). It has been previously reported that the 14-3-3η gene has a CRE binding site in its promoter (56) and as such its expression levels is likely to be regulated by changes in cellular levels of cAMP through the activation of the transcription factor CREB. Thus   AChRs + 14-3-3η (treated with forskolin), α4 S441A β2 AChRs and α4 S441A β2 AChRs +14-3-3η (treated with forskolin). The oocytes were clamped at a holding potential of -70mV.
ACh was applied successively following 4 min wash out periods following each application of ACh.  Transfected cells were fixed and processed for immunohistochemistry.