Function of Human α3β4α5 Nicotinic Acetylcholine Receptors Is Reduced by the α5(D398N) Variant*

Background: The naturally occurring α5(D398N) variant alters smoking behavior, but functional differences have not been detected between α3β4α5 nAChR harboring these variants. Results: ACh-induced α3β4α5 nAChR function is lower when α5(Asn-398) substitutes for α5(Asp-398). Conclusion: The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism. Significance: α3β4α5 nAChR function may be a useful target for smoking cessation pharmacotherapies. Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR α5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of α3β4α5 nAChR in Xenopus oocytes. α5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common α5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked α3, β4, and α5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric α3β4* nAChRs. α5 subunit incorporation reduces α3β4* nAChR function after coinjection with unlinked α3 and β4 subunits but increases that of α3β4α5 versus α3β4-only concatemers. α5 subunit incorporation into α3β4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of α5 subunits, free α3 and β4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit α3β4-only subtypes are dissimilar both to each other and to those of α3β4α5 nAChR. The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.

Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR ␣5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of ␣3␤4␣5 nAChR in Xenopus oocytes. ␣5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common ␣5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked ␣3, ␤4, and ␣5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric ␣3␤4* nAChRs. ␣5 subunit incorporation reduces ␣3␤4* nAChR function after coinjection with unlinked ␣3 and ␤4 subunits but increases that of ␣3␤4␣5 versus ␣3␤4-only concatemers. ␣5 subunit incorporation into ␣3␤4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of ␣5 subunits, free ␣3 and ␤4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit ␣3␤4-only subtypes are dissimilar both to each other and to those of ␣3␤4␣5 nAChR. The ␣5 variant-induced change in ␣3␤4␣5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.
Nicotinic acetylcholine receptors (nAChR) 2 are prototypical members of the ligand-gated ion channel superfamily of neu-rotransmitter receptors. nAChR exist as a diverse family of molecules composed of different pentameric combinations of homologous subunits derived from at least 17 genes (␣1-␣10, ␤1-␤4, ␥, ␦, ⑀). The properties of nAChR are determined by their subunit composition, giving rise to multiple subtypes with a range of overlapping pharmacological and biophysical properties (1). It also has become apparent that different stoichiometries of the same subunits can produce subtypes with distinctly different characteristics, a phenomenon observed in both heterologous and natural expression systems (1)(2)(3)(4)(5).
Recently, genome-wide association studies have indicated that single-nucleotide polymorphisms (SNPs) within nAChR subunits can substantially affect nAChR-mediated smoking behavior in humans. Most prominent among these single-nucleotide polymorphisms have been those located in the CHRNA5/CHRNA3/CHRNB4 locus, located on chromosome 15q25, which encodes the ␣5, ␣3 and ␤4 subunits of nicotinic receptors. This locus was first associated with nicotine dependence (6). Subsequent studies confirmed associations of singlenucleotide polymorphisms at this locus with heavy smoking (Ͼ25 cigarettes smoked daily), Fagerström Test for Nicotine Dependence scores and age dependent severity of nicotine dependence (7)(8)(9)(10)(11). One non-synonymous polymorphism (rs16969968), which changes the 398 th amino acid from aspartic acid to asparagine (D398N) in the ␣5 subunit, is particularly strongly associated with greater risk for increased nicotine consumption. Interestingly, variants at this locus also are associated with increased liability for lung cancer (8,12,13), and possibly with decreased risk for alcoholism (7,14) and cocaine dependence (15).
These observations raise the question of what the functional effects of the D398N mutation might be. The ␣5 subunit can only assemble into functional nAChR when expressed with at least two other subunits (1). In the central nervous system, most ␣5 subunit expression occurs in combination with ␣4 and ␤2 subunits (16,17). Experiments using heterologous expression systems have demonstrated that ␣4␤2* nAChR containing ␣5 subunits harboring the risk (Asn-398) variant have lower function than those that incorporate ␣5 subunits with the common (Asp-398) variant (11,18). This provides a mechanism through which the ␣5(D398N) mutation could produce phenotypic effects. Notably, a restricted set of brain regions (most prominently in the habenuolopeduncular pathway) express ␣5 subunits in combination with ␣3 and ␤4 subunits (19,20), as often occurs in autonomic ␣3␤4* nAChR (21)(22)(23). A recent study showed that increased expression of ␣3␤4* nAChR in the habenulopeduncular tract of mice increases nicotine aversion, an effect that can be reduced by the introduction and expression of additional ␣5(Asn-398) subunits in the same pathway (20). Furthermore, ␣3, ␤4, and ␣5 nAChR subunits are commonly expressed in bronchial, epithelial, and lung cancer cells, where nAChR activation by nicotine has been proposed as a mechanism that may increase tumor initiation and/or growth (24). However, heterologous expression studies done to date have not identified functional differences induced by ␣5 variant incorporation into ␣3␤4* nAChR (18,25).
Other observations may help to explain this discrepancy between in vitro observations and in vivo phenotypes. It has been shown that ␣3␤4 nAChR can be expressed in multiple stoichiometries, with different functional properties (26 -28). Moreover, ␣5 subunits can "compete" with ␤4 subunits for incorporation into assembled nAChR (29), possibly forcing formation of non-functional nAChR subunit assemblies as "dead end intermediates" (30). Thus, the effect(s) of common ␣5(Asp-398) versus risk ␣5(Asn-398) variant subunit incorporation into ␣3␤4* nAChR may be obscured by changes, attendant on any ␣5 subunit incorporation, in the overall level of ␣3␤4 nAChR functional expression and/or the balance of functional stoichiometric isoforms expressed. This complication in experimental interpretation is compounded when various mixtures of nAChR subtypes with specific subunit ratios are expressed from "loose" subunits assembled under host cell, and not investigator, control.
To overcome these difficulties in interpretation, we employed a concatemeric nAChR approach (Fig. 1). Here, nAChR constructs are assembled that encode all five subunits of the desired ␣3␤4* nAChR subtypes joined by short peptide linkers. The advantage of this approach is that complex nAChR subtypes can be expressed with native nAChR-like properties and with completely defined subunit ratios and orders of assembly (5,31). Using concatemeric ␣3␤4␣5 nAChR, we demonstrate that, as is true for ␣4␤2* nAChR, incorporation of the ␣5(Asn-398) variant reduces maximal acetylcholine-induced function when compared with the ␣5(Asp-398) variant. The properties of the defined concatemeric nAChR also were compared with those of ␣3␤4* nAChR allowed to assemble freely from loose individual subunits. These comparisons confirmed that concatemeric and freely assembled ␣3␤4␣5 nAChR have essentially indistinguishable pharmacological properties. Interestingly, these comparisons also suggested that loose ␣3 and ␤4 subunits associate quite differently in the presence or absence of ␣5 subunits.

EXPERIMENTAL PROCEDURES
Chemicals-All buffer components and pharmacological reagents (acetylcholine, atropine, cytisine, nicotine, and mecamylamine) were purchased from Sigma. Fresh stock drug solutions were made daily and diluted as required.
Constructs for Individual ␣3, ␤4, and ␣5 nAChR Subunits-Native human subunit protein sequences for ␣3, ␤4, and ␣5 (both Asn-398 and Asp-398 variants) nAChR subtypes were encoded by nucleotide sequences optimized for expression in vertebrate expression systems (synthesized by GeneArt AG; Invitrogen). Optimizations included minimization of high GC content sequence segments, improved codon usage, reduction of predicted RNA secondary structure formation, and removal of sequence repeats and possible alternative start and splice sites. Sequences were subcloned into the pSGEM oocyte high expression vector (a kind gift of Prof. Michael Hollmann; Ruhr-Universitaet, Bochum, Germany).
Concatemeric ␣3␤4 and ␣3␤4␣5 Constructs-Fully pentameric nAChR concatemers were constructed from human nAChR subunits. cDNAs encoding concatemers were created using the same subunit layout as successfully used to encode high and low agonist sensitivity ␣4␤2* nAChR isoforms (5). Subunits were arranged in the order ␤4-␣3-␤4-␣3-X, where X was either ␤4, ␣3, ␣5(Asp-398) or ␣5(Asn-398); Fig. 1A. Kozac and signal peptide sequences were removed from all subunit sequences with the exception of subunits expressed in the first position of the concatemer. As previously demonstrated, the initial ␤-␣ subunit protein pairs of the constructs will assemble to form an orthosteric binding site between the complementary (Ϫ) face of the initial ␤4 subunit and the principal (ϩ) face of the following ␣3 subunit (4). The assembled ␣3␤4* nAChR concatemers thus contain orthosteric agonist binding pockets at the ␤4(Ϫ)/(ϩ)␣3 interfaces between the first and second and between the third and fourth subunits (5). As for individual subunits, native human subunit protein sequences were encoded by nucleotide sequences optimized for expression in vertebrate expression systems (synthesized by GeneArt AG). Optimizations fell in the same categories as those previously described. Subunits were linked by alanine-glycine-serine (AGS) repeats designed to provide a complete linker length (including the C-terminal tail of the preceding subunit) of 40 Ϯ 2 amino acids. At the nucleotide level, linker sequences were designed to contain unique restriction sites that allow easy removal and replacement of individual ␣3, ␤4, and ␣5 subunits (Fig. 1A). Sequences of all subunits together with their associated partial linkers were confirmed by DNA sequencing (Geneart AG). Each concatemer was subcloned into the pSGEM oocyte high expression vector. Correct assembly of the concatemers into the expression vector was verified by restriction digest (Fig. 1, A and B). Additionally, concatemers were digested with ScaI to further diagnose the stoichiometry of each construct (* as indicated; Fig. 1B).
Oocyte Preparation and RNA Injection-Methods of oocyte isolation and processing for receptor expression have previously been described (32,33) but were modified as follows. Lobes were digested with 0.75 units/ml Liberase TM (Roche Applied Science), and oocytes were incubated at 13°C. The tips of pulled glass micropipettes were broken to achieve an outer diameter of ϳ40 m (resistance of 2-6 milliohms), and pipettes were used to inject 20 -60 nl containing 10 ng of cRNA/oocyte.
Data Analysis-EC 50 or IC 50 values and peak current amplitudes (I max ) were determined from individual oocytes. All stimulation protocols began with stimulation by a maximally efficacious dose of ACh (1 mM). This ensured that oocytes were indeed expressing functional nAChR before we did further recording, and it provided an internal control response for each oocyte. Relative agonist efficacies were calculated by comparison to this internal ACh control response. EC 50 and IC 50 values were determined through non-linear least squares curve-fitting (GraphPad Prism 4.0, GraphPad Software, Inc., La Jolla, CA) using unconstrained, monophasic logistic equations to fit all parameters, including Hill slopes. Additional normalization was used to compare absolute agonist efficacy between the concatemeric nAChR constructs. As for the nAChR expressed from loose subunits, all peak current response data were collected at 7 days post-injection. Function produced by oocytes expressing (␣3␤4) 2 ␣5(Asp-398) concatemers was chosen as the internal reference point for each batch of injected oocytes, as ␣5(Asp-398) is the more-common variant. Responses to 1 mM ACh, which is a maximally effective concentration for all of the constructs studied here, were measured. The mean function produced by oocytes injected with (␣3␤4) 2 ␣5(Asp-398) concatemers on each experimental day was used to normalize all of the data collected on that day. All four concatemeric constructs were tested in each experiment. In this way, any residual batchto-batch oocyte variation could be accounted for.
EC 50 and IC 50 values are presented as the mean Ϯ 95% confidence interval (CI). Data were analyzed using Student's t test to compare pairs of groups or by one-way or two-way ANOVA and Tukey's multiple comparison test to compare the means of three or more groups (PRISM, GraphPad Software, Inc.).
Only Intact nAChR Concatemers Contribute to Recorded Function-In some cases, the covalent linkers within concatemeric constructs have been observed to break down. This liberates smaller products that can assemble to form functional byproducts (4,34,35). To determine if this potential confound was present in our system, the ␣5(V 9Ј S) "gain-of-function" mutant was coinjected with either a concatemeric construct (␣3␤4)2␤4 or with individual ␣3 and ␤4 nAChR subunits. Assembly of the ␣5(V 9Ј S) subunit with either single subunits or abridged concatemers would result in a substantial gain of function (34,36). Co-expression of ␣5(V 9Ј S) with unlinked ␣3 and ␤4 subunits produced a significant increase in function (peak current amplitude elicited by 1 mM ACh; Fig. 4A). This demonstrates that the ␣5(V 9Ј S) subunit can assemble with nonlinked subunits as predicted. As previously noted, co-injection of a non-gain-of-function ␣5 subunit at a 1:1:1 ratio approximately halves ␣3␤4* function. This suggests that comparing nAChR function between oocytes injected with ␣3 and ␤4 subunits at a 1:1 ratio to that after injection with ␣3, ␤4, and ␣5(V 9Ј S) subunits at a 1:1:1 ratio may underestimate the effect of the gain-of-function mutation. In contrast, co-injection of the ␣5(V 9Ј S) subunit with the concatemeric construct, even at a 3:1 ␣5(V 9Ј S):concatemer ratio, produced no change in function (Fig. 4B). These data demonstrate that at least the great majority of nAChR function arising from injection of the concatemeric construct mRNAs must be mediated by intact, pentameric nAChR concatemers.
Absolute Efficacy Comparisons between (␣3␤4) 2 ␣5(Asp-398) and (␣3␤4) 2 ␣5(Asn-398) nAChR Concatemers-The studies above describe partial agonist efficacies normalized to ACh. However, we wanted to compare absolute agonist efficacies between constructs containing either the ␣5(Asp-398) or ␣5(Asn-398) variants. The use of concatemeric constructs allows these comparisons to be made without uncertainty related to the subunit makeup of the functional receptors. However, efficiency of functional nAChR expression varies across oocyte preparations and as a function of time post-injection. To compensate for this form of variation, we used a batchto-batch normalization strategy (described in detail under "Experimental Procedures").
constructs or after co-injection of single ␣3, ␤4, and ␣5 subunits (Table 1). We conclude that the presence of an ␣5 subunit or the use of concatemeric constructs results in the assembly of functional nAChR with similar pharmacological properties. These properties are likely the hallmark of assembly into a format containing two (␣3/␤4) subunit interfaces, with the addition of a fifth subunit in a non-ligand binding role. This conclusion is supported by a very recent study showing similar pharmacological profiles of (␣3␤4) 2 X nAChR assembled from ␣3-␤4 dimeric concatemers with the addition of single ␣3, ␤4, or ␣5 subunits (37). Without the constraints imposed by the concatemeric linkers or by the need to integrate a non-ligand binding ␣5 subunit, it seems possible that ␣3 and ␤4 subunits are free to assemble into at least two other formats. The relative proportions of the two formats expressed in the Xenopus oocyte system can be altered by biasing the ␣3:␤4 nAChR subunit mRNA injection ratio.

DISCUSSION
The pentameric concatemer approach allows accurate and consistent reproduction of complex nAChR subtypes, with complete control over subunit ratios and associations (5,31,38,39). It also allows for mutagenesis of a single subunit within an entire nAChR complex even where multiple copies of the target subunit may be present. These unique advantages were central to the work presented in this study. Using concatemeric ␣3␤4␣5 nAChR, we show that ␣5 subunit risk variant (Asn-398) incorporation reduces ACh-evoked function when compared with inclusion of the ␣5 common variant (Asp-398). Coexpression of unlinked ␣3, ␤4, and ␣5 subunits enforces assembly of an apparently uniform nAChR population with very similar pharmacological properties to those of concatemeric ␣3␤4* nAChR. In addition, either variant of the ␣5 subunit is capable of reducing the overall amount of ␣3␤4* nAChR function after coinjection with non-concatenated ␣3 and ␤4 subunits. Further observations suggested that removing the constraints imposed by either concatemerization or by co-expression with unlinked ␣5 subunits allows loose ␣3 and ␤4 subunits to assemble into at least two further subtypes. These ␣3␤4-only subtypes have substantially different pharmacological profiles from each other, from unlinked subunit ␣3␤4␣5 nAChR, and from any of the concatenated ␣3␤4 or ␣3␤4␣5 nAChR.
Critically, the pharmacological properties of ␣3␤4␣5 nAChR expressed using pentameric concatemers were similar to those of the same subtype expressed from unlinked subunits. This finding indicates that the addition of the concatemeric linkers did not noticeably alter nAChR function. It also reinforces further that pentameric concatemers faithfully replicate the ligand sensitivity of the equivalent subunit arrangement when formed from loose subunits. It has been suggested that ␣5 subunits compete with ␤4 subunits (20,29), reducing expression of functional ␣3␤4␣5 nAChR, possibly by encouraging the formation of dead-end intermediates that become trapped inside the cell (30). Our observations support this concept. Coinjection of non-concatenated ␣5 subunit mRNA approximately halved ␣3␤4* functional expression in Xenopus oocytes compared with injection of loose ␣3 and ␤4 subunits only (1:1:1 or 1:1 ratios were used; see the legend to Fig. 2). In contrast, if the ␣5 subunit is forced by concatemerization to assemble only as part of a pentameric nAChR complex, its incorporation substantially increases functional expression (Fig. 5). Together, these observations suggest that a reduction in function is not caused by the incorporation of ␣5 subunits per se. Instead, the presence of loose ␣5 subunits likely adversely affects the efficiency of unlinked ␣3 and ␤4 nAChR subunit assembly into functional nAChR.
In contrast, ␣3␤4-only nAChR expressed from pentameric concatemers had different pharmacological properties from those expressed from loose subunits ( Table 1, top two rows, and  Table 2). This discrepancy could be explained in several ways. One possibility is that covalent linkers may alter the properties of concatemeric nAChR by constraining structural transitions that are essential for normal function. This concern is mitigated by previous publications (5,31,38,39) indicating that well designed pentameric nAChR concatemers can accurately reproduce the properties of multiple native nAChR subtypes Maximal currents (normalized to (␣3␤4) 2 ␣5(Asp-398); see "Experimental Procedures") were compared between ␣3␤4* nAChR concatemers for acetylcholine (10 Ϫ6

␣5(D398N) Effects on ␣3␤4 Nicotinic Receptor Function
(which assemble from unlinked subunits). In addition, the linkers in each of the pentameric concatemers used in this study are of the same length and composition; it is unlikely that only the non-␣5* concatemers used in this study would suffer from linker-induced functional alterations. Furthermore, if the non-␣5* concatemers were uniquely affected by the presence of the linkers, it would be expected that this would strongly alter agonist potencies and relative efficacies when compared with those of the ␣5* concatemers. This is not the case; the pharmacological parameters measured from all four of the concatemers tested here are strikingly similar. A second possibility is that the covalent linkers within the concatemers might break down. This would release sub-pentameric products that could assemble to form unintended, but functional, byproducts (4,34,35). The presence of such degradation products was checked for by coinjection with an ␣5(V 9Ј S) mutant subunit. Assembly of this mutant subunit with either single ␣3 and ␤4 subunits or subpentameric concatemers would result in a substantial gain of function (34,36). No change in function was noted when ␣5(V 9Ј S) was co-injected with a concatemeric construct. This confirms that all, or nearly all, of the function in oocytes injected with pentameric nAChR mRNA constructs arises from fully-pentameric concatemeric nAChR. Finally, and most likely, the precise subunit associations imposed by concatemeric constructs may, or may not, correspond to those favored during association of loose subunits. Our data suggest that the ␣3␤4␣5 concatemers accurately reproduce the conformation adopted when the relevant individual subunits assemble freely. However, the same is not true for the ␣3␤4-only constructs when compared with nAChR assembled from loose ␣3 and ␤4 subunits. This would indicate that one role of the ␣5 subunit is to impose a particular subunit composition on ␣3␤4* nAChR expressed from loose subunits. If ␣5 is a true "accessory" subunit (i.e. does not interact directly with ligands), this may be unavoidable; a (␣3␤4) 2 ␣5 conformation is the only one in which two pairs of ␣3ϩ␤4 subunits would be available to provide agonist binding pockets and thus to assemble a functional ␣3␤4␣5 nAChR. The preceding observations raise the question of which nAChR subtype(s) is expressed after coinjection of only ␣3 and ␤4 subunits. This study confirms prior reports that at least two ␣3␤4 nAChR populations may be formed and that their relative expression levels depend on the molar injection ratio of the subunit mRNAs (1:20 versus 20:1). The pharmacology observed in this study matches that reported in other recent publications (27,28) that used less-extreme injection ratios (1:9 versus 9:1 or 1:10 versus 10:1). The lack of further changes in observed pharmacology after adoption of more extreme subunit ratios indicates that, as for ␣4 and ␤2 subunits (2,40,41), relatively pure populations of two different ␣3␤4 subunit assemblies are produced at the injection ratios used in this study. The same studies proposed again by analogy to the well-studied ␣4␤2 nAChR that the different nAChR isoforms might correspond to (␣3␤4) 2 ␤4 and (␣3␤4) 2 ␣3 nAChR (27).
Accordingly we constructed (␣3␤4) 2 ␤4 and (␣3␤4) 2 ␣3 concatemers using the same subunit arrangements as used successfully to encode high and low agonist sensitivity pentameric ␣4␤2 nAChR concatemers (5). We initially anticipated that these concatemers would have similar pharmacological profiles to ␣3␤4-only nAChR formed after injection of loose ␣3 and ␤4 subunits at 1:20 and 20:1 ratios, respectively. However, the pharmacology observed after injection of loose ␣3 and ␤4 subunits at either 1:20 or 20:1 ratios (Fig. 6, Table 2) was strikingly different from the concatemeric "(␣3␤4) 2 X-type" measurements. The precise arrangements adopted by loose ␣3 and ␤4 subunits injected at different ratios remain unknown. It certainly seems probable that 1:20 and 20:1 ␣3:␤4 injection ratios may give rise to nAChR with different stoichiometries (27,28). In addition, as demonstrated for GABA A receptors, the precise order of subunit incorporation (even for identical subunit stoichiometries) can affect receptor function (42). The emerging awareness that agonist binding to non-canonical nAChR interfaces can strongly affect function underlines this point (5,41,43). Determining whether different ␣3:␤4 subunit mRNA injection ratios produce nAChR with different stoichiometries, different arrangements of the same subunit stoichiometries, or both will require a great deal more investigation. The concatemeric pentamer approach is uniquely well suited to addressing this question.
Unlike agonist EC 50 values, IC 50 values for mecamylamine inhibition were greatly affected by the identity of the fifth subunit in each pentameric concatemer. This suggests that mecamylamine (a non-competitive antagonist) interacts with the resulting nAChR in a position where it can be influenced by the presence of alternate subunits in the fifth, non-agonistbinding position. This sensitivity to ␣3␤4* nAChR composition was also evident when comparing mecamylamine IC 50 values between ␣3␤4 and ␣3␤4␣5 nAChR expressed from loose subunits (Table 1). These observations indicate that noncompetitive ligands may provide the best opportunities to pharmacologically distinguish between different subunit arrangements of ␣3␤4* isoforms. Importantly, this category could also include positive allosteric modulators and/or allosteric agonists in addition to non-competitive antagonists. Given the association of ␣5 subunit variants with a variety of substance abuse behaviors (see introduction), selective manipulation of ␣3␤4␣5 nAChR activity could have valuable therapeutic implications.
It appears that, as previously proposed (20), ␣5 subunit expression may act to modulate the amount of ␣3␤4* nAChR function in the habenulopeduncular tract and in other tissues that express ␣3␤4␣5 nAChR. This study indicates that the presence of the ␣5(Asp-398) or ␣5(Asn-398) variant will impose an additional layer of functional modulation. As noted previously (18), the concentrations of nicotine present in smokers are too low to significantly activate or desensitize ␣3␤4␣5 nAChR. However, the activity induced by synaptic or perisynaptic ACh release onto ␣3␤4* nAChR could be strongly affected by the integration of ␣5(Asp-398) or ␣5(Asn-398) subunits. This in turn could result in compensatory changes either at the neurotransmitter/receptor level or at the circuit activity level, which may explain some of the phenotypic variations attributed to the ␣5(D398N) mutation. Given the established role of the habenulopeduncular pathway ␣3␤4␣5 nAChR function in nicotine dependence and aversive behavior (20,44,45), it seems likely that selective manipulation of ␣3␤4␣5 function mediated by this subtype could represent a valuable smoking cessation strategy. Our current findings indicate that non-competitive/ allosteric compounds may be the most promising category of potential therapeutic agents for such an approach.