Unorthodox Acetylcholine Binding Sites Formed by α5 and β3 Accessory Subunits in α4β2* Nicotinic Acetylcholine Receptors*

All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric α4β2* nAChRs typically have two ACh binding sites at α4/β2 interfaces and a fifth accessory subunit surrounding the central cation channel. β2 accessory subunits do not form ACh binding sites, but α4 accessory subunits do at the α4/α4 interface in (α4β2)2α4 nAChRs. α5 and β3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that α5/α4 and β3/α4 interfaces formed ACh binding sites in (α4β2)2α5 and (α4β2)2β3 nAChRs. The α4/α5 interface in (β2α4)2α5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30–50%. However, site-selective agonists NS9283 (for the α4/α4 site) and sazetidine-A (for the α4/β2 site) did not act on the ACh sites formed by the α5/α4 or β3/α4 interfaces. This suggests that unorthodox sites formed by α5 and β3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders.

Nicotinic acetylcholine receptors (nAChRs) 2 are ACh-gated ion channels formed from five homologous subunits organized like barrel staves around a central cation channel (1). There are homomeric nAChRs and heteromeric nAChRs. Homomeric ␣7 nAChRs have five ␣7 subunits with ACh binding sites between each of the five ␣7/␣7 interfaces. All nAChR subunits exhibit basic homology throughout their sequences, indicating that all nAChRs evolved from a common ancestor (1,2). Heteromeric nAChR agonist binding sites typically form at the interface between the primary (ϩ) side of an ␣ subunit, characterized by the presence of a C loop that closes over the site when the nAChR is activated by an agonist, and the complementary (Ϫ) side of a ␤2 or ␤4 subunit (3). Two such ACh binding sites assemble with an accessory subunit in a stoichiometry such as (␣4␤2) 2 ␤2 (4, 5).
The (␣4␤2) 2 ␣4 stoichiometry contains an unorthodox ACh binding site at the ␣4/␣4 interface (6,7). When this low affinity unorthodox site is bound by ACh or NS9283, an agonist specific for this site, nAChRs activate 3-4-fold more efficiently (8,9). Blocking the ␣4/␣4 site through alkylation of a cysteine introduced in the minus face of the ␣4 subunit blocked the activity of ACh and NS9283 (9). Histidine 142 (this is position 116 in the mature ␣4 peptide sequence) on the minus side of ␣4 is critical for the binding of NS9283 (10). Sazetidine-A is a high affinity agonist selective for ␣4/␤2 sites that cannot bind to the ␣4/␣4 interface because histidine 142 on the minus side of ␣4 prevents it from binding, whereas this amino acid is valine in ␤2 (11,12). Because orthodox agonist sites are shared among nAChRs, subtype selectivity is more likely to be achieved by targeting unorthodox agonist sites.

Model Depicting nAChRs Formed by Concatamers-To study
ACh homologue sites formed by ␣4 with ␣5 or ␤3 subunits, we constructed these subunit interfaces using concatamers with ␣4 and ␤2 subunits linked in different orders (Fig. 1). These illustrations are important for understanding the different concatamers used to form desired interfaces, such as the ␣5/␣4 interface versus the ␣4/␣5 interface. To represent the assembled orientation of linked subunits, nAChRs assembled from ␤2-␣4 and a free subunit are noted as (␣4␤2) 2 *. Correspondingly, (␤2␣4) 2 * represents nAChRs assembled from a ␣4-␤2 concatamer. Fig. 1A illustrates how ␤2-␣4 dimeric concatamer plus free ␣5 or ␤3 subunit is thought to assemble into nAChRs. Concatamers are generated by connecting the C-tail of one subunit to the N terminus of the next. The order of the subunits around the cation channel can be determined by the length of the linker between the subunits (30). When the 23-amino acid-long C-terminal tail of ␤2 subunits is linked by (AGS) 6 , or a longer linker to the N terminus of ␣4, a binding site forms within the linked subunit pair to form an ␣4/␤2 ACh binding site (30). This allows the free subunit to assemble as an accessory subunit in combination with two ␣4/␤2 ACh binding sites. Fig. 1B illustrates how ␣4-␤2 dimeric concatamer plus free ␣5 subunit is thought to assemble into nAChRs. The linker of ␣4-␤2 dimeric concatamer is much shorter than that of the ␤2-␣4 dimeric concatamer. When the short 7-amino acid C-tail of ␣4, compared with the 23-amino acid-long C-tail of ␤2, is linked by (AGS) 6 to the N terminus of ␤2, the subunits are constrained to assemble so that ␣4/␤2 ACh binding sites are formed between linked pairs of subunits, and ␣5 assembles with the plus rather than minus face of ␣4 to form an ␣4/␣5 interface.
Immunoblots of Triton X-100 extracts from oocytes injected with concatamers confirmed the integrity of expressed proteins (Fig. 2). Concatamer ␤-6-␣ and ␣-6-␤ migrated at a molecular mass of ϳ118 kDa. These data demonstrated that concatamer cRNAs were properly translated in the oocytes, and no degradation to unlinked subunits was seen.
Cysteine Mutants Used to Study Specific Interfaces Formed by ␤3 and ␣5 Subunits-Because sazetidine-A is a partial agonist for (␣4␤2) 2 ␣5, (␣4␤2) 2 ␤3, and (␤2␣4) 2 ␣5 nAChRs, it is likely that sazetidine-A only binds to the ␣4/␤2 interfaces, but ACh binds to both the ␣4/␤2 and additional sites formed by ␤3 or ␣5 subunits (Fig. 1). To test this hypothesis and evaluate which interfaces forms ACh sites, we mutated a cysteine at the minus site of various subunit interfaces (Figs. 5 and 6 and Table 2). Alkylation of this cysteine residue will block the ACh homologue sites in the corresponding subunit interfaces (9, 33). If this interface forms an agonist site, MTSET will block the activation of ACh. No change of response is expected if MTSET reacts with the cysteine at interfaces that do not bind an agonist (Figs. 5 and 6) (9, 33).
The ␣5/␣4 and ␤3/␣4 interfaces can be selectively blocked by mutating threonine 126 on the minus face of the ␣4 subunit to a cysteine residue followed by alkylation of the cysteine with MTSET. The ␤2/␣5 and ␤2/␤3 sites were investigated by mutating the threonine 139 residue on the minus face of the ␣5 subunit and mutating the threonine 123 residue on the minus face of the ␤3 subunit. The ␣5 subunit contains a free cysteine at position 2. To prevent nonspecific modification or any potential disulfide formations between the single free cysteine at position 2, we mutated the cysteine to a serine (33). The mutations did not significantly change the level of expression. Maximum responses evoked by ACh were similar among WT and mutants: The concentration of MTSET used for alkylation experiments was 0.5 mM for ␣5 containing nAChRs because higher concentrations resulted in a significant decrease in the ACh response of wild type nAChRs. The concentration of MTSET used for alkylation experiments for ␤3 containing nAChRs was 2 mM, the concentration at which the wild type response was not affected after alkylation. With 5 mM MTSET, blockage (ϳ30%) was similar to that with 2 mM. . Schematic illustration of alkylation of (␣4␤2) 2 ␣5 nAChRs. nAChRs were expressed from wild type or mutant ␤2-␣4 dimeric concatamers co-expressed with wild type or mutant ␣5 subunits. The ␣4 T126C mutation was introduced to provide a cysteine at the minus face of the ␣5/␣4 interface, and the ␣5 T139C mutation was introduced to provide a cysteine at the minus face of the ␤2/␣5 interface, where alkylation of the cysteine by MTSET allowed for the selective blockage of the desired interface. The free cysteine at position 2 of ␣5 was mutated to a cysteine-null pseudo wild type ␣5 C2S . NOVEMBER 4, 2016 • VOLUME 291 • NUMBER 45
When ␣5 C2S was co-expressed with the ␤2-␣4 T126C dimeric concatamer, there was no additional blockage by MTSET compared with using wild type ␣5, indicating that the block observed in the wild type ␣5 was solely due to blockage of activation by ACh at the ␣5/␣4 interface (Fig. 7D).
This suggests that (␣4␤2) 2 ␣5 nAChRs contain three functional ACh binding sites: two within the dimeric concatamers at the two ␣4/␤2 interfaces and one at the ␣5/␣4 interface. All the values for the ACh concentration/response curves before and after MTSET alkylation for ␣5 containing nAChRs are presented in Table 3.
Wild type nAChRs were tested before and after MTSET treatment to demonstrate that there was no block in response and to provide a comparison with the mutant nAChRs (Fig. 8A). Incubation of oocytes with MTSET for 5 min reduced activation by ACh for (␣4 T126C ␤2) 2 ␤3 nAChRs by ϳ25-30% of the original ACh response (p ϭ 0.0061) (Fig. 8, B and C). However, there was no block of response to a saturating (100 nM) concentration of sazetidine-A in this mutant (Fig. 8B). This confirms the idea that sazetidine-A can bind to and activate the ␣4/␤2 sites but not ␤3/␣4 sites.
In addition to the ␤3/␣4 interface, we also investigated whether the ␤2/␤3 interface formed a functional ACh binding site by mutating threonine 123 at the minus face of ␤3 to a cysteine. Unlike the ␤3/␣4 interface, the ␤2/␤3 interface did not form an ACh binding site. When ␤3 T123C was co-expressed with the ␤2-␣4 dimeric concatamer, the response after alkylation was similar to the wild type subtype (Fig. 8D).
This suggests that (␣4␤2) 2 ␤3 nAChRs contain three functional ACh binding sites: two within the dimeric concatamers at the two ␣4/␤2 interfaces and one at the ␤3/␣4 interface. The potencies and efficacies of ACh before and after MTSET alkylation for ␤3 containing nAChRs are presented in Table 4.
MTSET blocks activation from the ␣4/␣5 site, reducing the response to 45.80 Ϯ 0.05% after alkylation (Fig. 9B). These nAChRs are thought to have one conventional agonist site at FIGURE 6. Schematic illustration of alkylation of (␣4␤2) 2 ␤3 nAChRs. nAChRs were expressed from wild type or mutant ␤2-␣4 dimeric concatamers co-expressed with wild type or mutant ␤3 subunits. The ␣4 T126C mutation was introduced to provide a cysteine at the minus face of the ␤3/␣4 interface, and the ␤3 T123C mutation was introduced to provide a cysteine at the minus face of the ␤2/␤3 interface, where alkylation of the cysteine allowed for the selective blockage of the desired interface by MTSET.

TABLE 2 Mutants used for MTSET alkylation experiments
Locations of mutations are underlined. Binding site mutations are in loop E on the minus side of the site. The C2S mutation is near the N terminus.

Mutant
Location of mutations the ␣4/␤2 interface and one unorthodox site at the ␣4/␣5 interface that can be blocked by MTSET. Therefore any decrease in response seen after MTSET treatment of (␤2␣4) 2 ␣5 nAChRs can be attributed to specific block of the ␣4/␣5 interface. MTSET only blocked ϳ54% of maximal response of (␤2␣4) 2 ␣5 nAChRs. This remaining response could result from activation from the single conventional ACh site in the ␣4/␤2 interface or inefficient blockage of the ␣4/␣5 site.

TABLE 3 Summary of potencies and efficacies of ACh activating wild type and mutant (␣4␤2) 2 ␣5 nAChRs before and after MTSET treatment
The nAChRs are expressed from concatamer ␤2-␣4 and free ␣5 subunit in Xenopus oocytes. Efficacy indicates the maximum efficacy relative to ACh before MTSET treatment. n indicates the number of oocytes tested.

␤2-␣4 ؉ ␣5
␤2-␣4 T126C ؉ ␣5 cysteine, as previously described (9). When both of the ␣4/␤2 sites were blocked with MTSET, blockage of response was essentially complete (Fig. 9). This indicates that one ␣4/␤2 was able to sufficiently activate the channel by itself if another functional binding site was deactivated, but when both of the ␣4/␤2 sites were deactivated, the channel was not able to open with only the ␣4/␣4 binding site. Therefore, the remaining response seen in MTSET-treated (␤2␣4) 2 ␣5 nAChRs is likely from activation from the single conventional ACh site in the ␣4/␤2 interface.
Presence of Pentameric ␣5␤2 Assemblies without Functional ACh Binding Sites-Because MTSET blocks activation from ␣5/␣4 and ␣4/␣5 sites, ␣5 can assemble like a conventional ␣4 or ␤2 subunit to form an ACh binding site. We showed that the ␤2/␣5 site did not form an ACh site (Figs. 5 and 7E). To investigate whether ␣5/␤2 forms an ACh binding site, we injected oocytes with free ␤2 and ␣5. Any response detected should result from activation of ␣5/␤2 site.
We first confirmed that ␣5 and ␤2 assembles into pentameric nAChRs using sucrose gradient sedimentation (Fig. 10A). These sucrose gradient fractions were immunoisolated by mAb210 (against ␣5 subunit) and detected by 125 I-mAb 295 (against ␤2 subunit) to study assembly of nAChRs containing both ␤2 and ␣5 subunits. ␤2 and ␣5 monomeric subunits assembled to form aggregates that sedimented at nearly the size of 9.5S Torpedo nAChR monomers and 13S dimers. Aggregates at the size of 13S dimers of Torpedo nAChRs suggest the presence of dipentamers (Fig. 10B). Dipentamers might form through disulfide linking of cysteines near the N terminus of ␣5. Torpedo nAChR dipentamers are linked by disulfide bonds between cysteines at the C terminus of ␦ subunits (37).

Discussion
Discovery of an ␣4/␣4 interface that formed an unorthodox ACh binding site that greatly potentiated the effect of the two orthodox ␣4/␤2 ACh binding sites and is a target for the siteselective agonist drug NS9283 altered our understanding of how nAChRs work (6,9). Here we show that ␣5 and ␤3 subunits, which were previously not thought to form ACh binding sites, can form ␣5/␣4, ␤3/␣4, and ␣4/␣5 ACh binding sites. The ␣5/␣4 ACh binding site increases the response from (␣4␤2) 2 ␣5 nAChRs almost 2-fold. ␣5 subunit greatly increases the Ca 2ϩ permeability of (␣4␤2) 2 ␣5 nAChRs (38). Increased response and high Ca 2ϩ permeability caused by the presence of ␣5 makes the ␣5/␣4 ACh binding site on this nAChR subtype an appealing drug target. Unorthodox sites may be useful for targeting drugs that can be exceptionally specific for important nAChR subtypes. The unique binding sites at the ␣5/␣4 and ␤3/␣4 interfaces are sites at which ACh site-selective agonists might act, much as NS9283 is able to act as an ACh site-selective agonist at the unique ␣4/␣4 interfaces of (␣4␤2) 2 ␣4 nAChRs to potentiate their function (8,9).
A previous study concluded that a functional ␣5/␣4 binding site was not present in mouse (␣4␤2) 2 ␣5 nAChRs (39). In their studies, when the aromatic box residues in ␣5 were mutated to inhibit the interaction with the quaternary amine of ACh in ␣5, they observed no change in EC 50 and concluded that there is no ACh binding site present at the ␣5/␣4 interface. However, these mutations did cause a large decrease in the amplitude of response, which is consistent with our findings that the response is potentiated by a third ACh site present at the ␣5/␣4 interface.
It has previously been reported that ␣4/␣5 may form a functional ACh binding site (40). Our results are consistent with this observation.
In conclusion, unorthodox ACh binding sites can form at the ␣5/␣4 and ␤3/␣4 interfaces in (␣4␤2) 2 ␣5 and (␣4␤2) 2 ␤3 nAChRs and at the ␣4/␣5 in (␤2␣4) 2 ␣5 nAChRs. Looking forward, these unique interfaces can be targets for unique drugs, specifically site-selective agonists to bind to them for potential treatment of diseases or nicotine addiction. These unorthodox sites provide reason to believe that unorthodox sites might also exist at ␤3/␣6 interfaces. This site would provide a specific target on the complex (␣6␤2)(␣4␤2)␤3 nAChR subtype that is important in regulating dopamine release and potentially important in smoking cessation therapy and other applications associated with learning and motor control (15,16). The synthesis of specific drugs that can selectively bind to these unorthodox sites might play a significant role in the treatment of nicotine addiction through activation of (␣4␤2) 2 ␣5 (29) or antagonism of (␣6␤2)(␣4␤2)␤3 nAChRs (18).

Experimental Procedures
Chemicals-MTSET was purchased from Toronto Research Chemicals Inc. (North York, Canada). NS9283 was synthesized as described (42). 25 and 10 mM stocks of NS9283 were prepared in DMSO. MTSET solutions were freshly prepared from solid daily and kept on ice until used. Dilutions of all drugs were prepared daily in ND96 testing buffer before use. All other chemicals were purchased from Sigma-Aldrich unless otherwise noted.
Point mutations to various subunits were introduced using the PfuUltra high fidelity DNA polymerase (Agilent, Santa Clara, CA) or the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla CA), following the manufacturer's instructions. Two separate point mutations were introduced in ␣5. A threonine to cysteine mutation at position 139 was introduced to allow alkylation of ACh site at minus face of ␣5 subunit. To prevent nonspecific modification or any potential disulfide formations between the single free cysteine at position 2 of ␣5, this cysteine was mutated to serine. One point mutation was introduced in ␤3. The threonine present at position 123 in the minus side of ␤3 was mutated to a cysteine. Mutated dimeric concatamers, ␤2 L121C -(AGS) 6 -␣4 (abbreviated ␤2 L121C -␣4) and ␤2-(AGS) 6 -␣4 T126C (abbreviated ␤2-␣4 T126C ), were prepared as described (9). All oligonucleotides and the amino acids mutated are listed in Table 2. The mature amino acid sequences were used to number nAChR subunits.
To prepare the ␤2 L121C -␣4-␤2-␣4 tetrameric concatamer, the ␤2 L121C -␣4 dimeric concatamer and the ␤2-␣4-␤2 trimeric concatamer were cut by BsiWI restriction enzyme unique to ␤2 subunit. The 3390-bp BsiWI fragment from ␤2-␣4-␤2 trimeric concatamer was inserted into ␤2 L121C -␣4 concatamer using T4 DNA ligase (New England Biolabs). The correct clone was cho- FIGURE 10. Sucrose gradient sedimentation and surface expression of oocytes injected with free ␤2 ؉ free ␣5. A, sucrose gradient sedimentation of Triton X-100 extract from oocytes injected with ␤2 and ␣5. Components containing ␣5 subunits of the 63 sucrose fractions were immunoisolated using mAb 210-coated wells. ␣5␤2 nAChRs in these wells were then quantified using I 125 -mAb 295 (against ␤2 subunit). An internal standard in the gradient was Torpedo nAChR that was resolved into 9.5S monomers and 13S dimers. Arrows indicate the locations of the monomer and dimer peaks of Torpedo nAChRs. The gradient revealed that the wild type ␤2 and ␣5 free subunits associated to form aggregates that sedimented at nearly the size of 9.5S Torpedo nAChR monomers and 13S dimers of Torpedo nAChRs. B, surface expression of nAChRs in oocytes injected with ␤2 and ␣5 subunits. Radioactively labeled 125 I-mAb 295 to ␤2 subunit was used to determine surface expression of nAChRs, and uninjected oocytes were used to determine nonspecific binding.
sen from the one that contained two fragments, 3400 and 7100 bp, after digestion by BstEII restriction enzyme (unique for ␣4).
Xenopus Oocyte Preparation-Oocytes were harvested from Xenopus laevis frogs in accordance to the approved institutional animal care and use committee protocol as described (9,46). The oocytes were washed three times with OR2 (85.5 mM NaCl, 2 mM KCl, 1 mM MgCL 2 , and 5 mM HEPES, pH 7.5) solution and three times with 50% Leibovitz-15 (L-15) medium (Invitrogen), 10 mM HEPES, pH 7.5, 10 units/ml penicillin, 10 g/ml streptomycin. The oocytes were then placed in Leibovitz-15 medium for a couple hours to allow for recovery before injection.
cRNA Microinjection-Subunit cRNAs were mixed in different ratios to produce the desired constructs. Proper ratios were determined by performing expression tests with ACh only and ACh coappplied with 10 M NS9283. If potentiation was seen with NS9283, this indicated the presence of dipentamers and the ratio was adjusted until no potentiation was seen. To obtain (␣4␤2) 2 ␣5 nAChRs, oocytes were injected with a total of 5-10 ng of cRNA, two parts of ␤2-␣4 to one part of free ␣5. To obtain (␣4␤2) 2 ␤3 nAChRs, the oocytes were injected with a total of 90 ng of cRNA, one part of ␤2-␣4 to two parts of free ␤3. To obtain (␤2␣4) 2 ␣5 nAChRs, the oocytes were injected with 150 ng of cRNA, six parts of ␣4-␤2 to one part of free ␣5. To obtain (␣4␤2␣4␤2)␤2 nAChRs, the oocytes were injected with 30 ng of cRNA, two parts of ␤2-␣4-␤2-␣4 and one part of ␤2. For surface labeling assays, 60 ng of free ␤2 and free ␣5 was injected at a one to one ratio. These cRNA ratios are indicated in terms of weight. Because cysteine mutations in this study did not change protein expression, the cRNA ratios of mutants used were the same as wild type. After microinjection, the oocytes were incubated in medium made up of L-15 with 50 g/ml gentamycin and switched out to L-15 without gentamycin before electrophysiological recording. Function was assayed 3-7 days after injection.
Surface Labeling-Radioactively labeled 125 I-mAb 295 to ␤2 was used to determine surface expression of the nAChRs (47). Surface labeling was performed on the same day as electrophysiological recording. Each set of oocytes, injected with different constructs, was placed in separate Eppendorf tubes with a total volume of 500 l of L-15 medium containing 3% bovine serum albumin with 5 nM 125 I-mAb 295 at room temperature for a minimum of 3 h (47). The oocytes were washed three times to remove unbound 125 I-mAb 295 to ␤2 or until there was no change in activity measured by a Geiger counter. The oocytes were put into individual tubes, and bound 125 I was measured using a ␥-counter. Uninjected oocytes were used as the control for nonspecific binding.
Sucrose Gradient Sedimentation-A group of 60 oocytes was homogenized in 1 ml of buffer A (50 mM Na 2 HPO 4 , pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride) and then pelleted by centrifuging at 13,400 rpm for 15 min at 4°C. The pellets were resuspended by pipetting, and membrane proteins were solubilized in 150 l of buffer A containing 2% Triton X-100 for 1 h at room temperature on a rotator. Debris was removed by centrifugation at 13,400 rpm for 15 min at 4°C. Aliquots (150 l) of the lysates, mixed with 1 l of 2 mg/ml purified Torpedo californica electric organ nAChR, were loaded onto 11.4 ml sucrose gradients (linear 5-20% sucrose (w/w) in 10 mM sodium phosphate buffer, pH 7.5, that contained 100 mM NaCl, 1 mM NaN 3 , 5 mM EDTA, 5 mM EGTA, and 0.5% Triton X-100). Gradients were centrifuged for 16 h at 40,000 rpm in a SW-41 rotor (Beckman Coulter, Fullerton, CA) at 4°C. Fractions were collected at 15 drops/well from the bottom of the tubes and used for additional analysis. Then 50 l of each fraction were transferred to mAb 210-coated wells to isolate ␣5-containing nAChRs and incubated with 5 nM 125 I-mAb 295 to detect ␤2-containing nAChRs. Another 20 l of each fraction were transferred to mAb 210-coated wells incubated with 1 nM 125 I-␣-bungarotoxin overnight at 4°C to isolate and detect ␣1-containing T. californica nAChRs, which are used as molecular mass standards. Afterward, the wells were washed three times with PBS and 0.5% Triton X-100, and bound 125 I-mAb 295 and 125 I-␣-bungarotoxin were determined by ␥-counting.
Solid Phase Radioimmunoassay-Total epibatidine binding was determined using an increasing amount of extract loaded with 1 nM [ 3 H]epibatidine (PerkinElmer Life Sciences) in a total volume of 100 l in PBS buffer containing 0.5% Triton X-100 and 10 mM NaN 3 (47). Binding took place on a horizontal rotator for a minimum of 2 h at room temperature or overnight at 4°C. After incubation, the wells were washed three times with 0.5% Triton X-100 in PBS before elution with 30 l of 0.1 M NaOH solution. Bound radioactivity was determined using a 1450 Trilux Microbeta liquid scintillation counter (Perkin-Elmer Life Sciences) with OptiPhase scintillation fluid as described (47). Nonspecific binding was determined using uninjected oocytes.
Electrophysiology-Functions of nAChRs were measured by two-electrode voltage clamp using either a manual two-microelectrode voltage clamp amplifier setup (oocyte clamp OC-725; Warner Instrument, Hamden, CT) or OpusXpress 6000A (Molecular Devices, Union City, CA), an integrated system that provides automated impalement, voltage clamp, and simultaneous drug delivery to eight oocytes in parallel (48) (49). Voltage was held at Ϫ50 mV.
In the agonist concentration/response experiments, the oocytes first received two control applications of 300 M ACh followed by applications of increasing concentrations of agonists. The peak amplitudes were normalized to the average of the maximum ACh response evoked by initial two controls.