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. Author manuscript; available in PMC: 2016 Oct 21.
Published in final edited form as: Trends Pharmacol Sci. 2016 May 11;37(7):562–574. doi: 10.1016/j.tips.2016.03.005

Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain

Jie Wu 1,2,*, Qiang Liu 2, Pei Tang 3, Jens D Mikkelsen 4, Jianxin Shen 1, Paul Whiteaker 2, Jerrel L Yakel 5
PMCID: PMC5074342  NIHMSID: NIHMS821668  PMID: 27179601

Abstract

The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling.

Traditional Rule of Assembly of nAChRs

Nicotinic acetylcholine receptors (nAChRs) in mammals exist as a diverse family of channels composed of different combinations of subunits derived from at least 17 genes [1,2]. Functional nAChRs can be assembled as both heteromers containing α and β subunits, or as homomers containing only α subunits [1,2]. In the mammalian brain, the most abundant forms of nAChRs are the heteromeric α4β2 nAChR and the homomeric α7 nAChR [3–7]. Homomeric α7 nAChRs are assembled with 5 α7 subunits, which are distinguished from the majority of other nAChR subtypes that require different subunit assembly partners (e.g., α plus β subunits). Naturally expressed α7 nAChRs might mediate classic excitatory neurotransmission at some loci, but they may also modulate the release of other neurotransmitters, neurite outgrowth, and even neuronal survival/death [8,9]. In addition, α7 nAChRs express not only on neurons but also on various types of non-neuronal cells including astrocytes and microglia [10,11] (Box 1). The α7 nAChR, as a target of nicotine action, also has been implicated in the development, differentiation, and pathophysiology of the nervous system [12,13]. In rat forebrain cholinergic neurons, α7 and β2 are the predominant nAChR subunits, and they were found to colocalize [14]. Accumulating evidence demonstrates that these α7 and β2 nAChR subunits can co-assemble to form functional α7β2 nAChRs in heterologous expression systems, and also under natural conditions in the brain (Figure 1) [15,16]. Importantly, this novel type of heteromeric α7β2 nAChR exhibits unique function, pharmacology, and pathophysiology, and likely is an important target for cholinergic modulation in neuronal function and disease.

Box 1. α7 nAChRs Express in Non-Neuronal Cells.

The α7 nAChRs are widely expressed in the central and peripheral nervous systems, in neurons as well as in several nonneuronal cells. For example, emerging evidence demonstrates that α7 nAChRs are expressed in immune cells including macrophages/monocytes [56], dendritic cells [57], microglia [58] and astrocytes [10,11]. When the α7 nAChRs are activated, nicotine exhibits significant modulations in the production of proinflammatory cytokines, such as interleukin (IL)-1, TNF-α, and IL-1β [59]. Therefore, the α7 nAChRs in the immune system play an important role in the regulation of multiple immune pathways and inflammatory responses [60,61]. The α7 nAChRs are also expressed in stem cells, where they likely mediate cholinergic signaling on these cells and regulate survival/apoptosis, proliferation, differentiation, and maturation [62,63]. Lastly, α7 nAChRs are also expressed in endothelial cells, bronchial epithelial cells, skin keratinocytes, and vascular smooth muscle cells, where these nAChRs are likely involved in the regulation of the function and pathology of these cells [64–66].

Figure 1.

Figure 1

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Cartoon Figure Showing Homomeric and Heteromeric α7 Nicotinic Acetylcholine Receptors (nAChRs). Yellow dots indicate the α7–α7 interface for ligand binding sites, blue dots the α7–β2 interface, the orange dot indicates the β2–β2 interface, and black dots the β2–α7 interface. It is important to note that the subunit ratio (s) and subunit–subunit associations within native α7β2 nAChR subtypes are not yet known, although studies using heterologous expression systems indicate that multiple arrangements are compatible with α7β2 nAChR function.

What is a heteromeric α7 nAChR?

Traditionally, neuronal nAChRs are classified into two subgroups based on their subunit combination: the heteromeric α and β pentamers (such as the α4β2 nAChR), and the homomeric α pentamers (such as the α7 nAChR) (Figure 1). Here, the heteromeric α7 nAChR is defined as α7 nAChR subunits that are co-assembled with the β2 nAChR subunit(s) to form a novel type of α7β2 nAChR (Box 2).

Box 2. Heteromeric and Homomeric nAChRs.

nAChRs are prototypical members of the cys-loop ligand-gated ion channel superfamily of neurotransmitter receptors. Neuronal nAChRs are found throughout the nervous system (e.g., in muscle, autonomic and sensory ganglia, and the CNS). nAChRs exist as multiple, diverse subtypes composed as pentamers of unique combinations from a family of at least seventeen (α1–α10, β1–β4, δ, δ, ε) similar, but genetically distinct, subunits [1]. There are two classic groups of nAChRs based on the pattern of their subunit combinations. Most of these nAChR subtypes appear to exist as heteropentamers containing two or more different types of subunits, called heteromeric nAChRs. For example, heterologous expression studies suggest that α2, α3, α4, or α6 subunits can combine in binary fashion with β2 or β4 subunits to form ligand-binding and/or functional nAChRs (e.g., the α4β2 nAChR is abundantly expressed naturally in the brain) or in tertiary complexes containing more than one of these α or β subunit types. Another prominent nAChR subtype found in vertebrate central and autonomic nervous systems contains unique α7 subunits (α7 nAChR), called homomeric nAChRs. These two groups of nAChRs exhibit distinct function, pharmacology, and pathophysiology.

Evidence of Heteromeric α7β2 nAChRs

The existence of heteromeric α7β2 nAChRs has been shown in both heterologously expressed systems and native brain neurons by multiple experimental approaches. Early studies from Lindstrom and colleagues have demonstrated that chick retinal ganglion neurons express heteromeric α7 and α8 nAChRs that may include other α and/or β nAChR subunits [17–19]. Biochemical studies also support this possibility because the α7 protein can be immunoprecipitated with other subunits, although the identity of these subunits was not determined at that time[20]. Using a presynaptic preparation, McGehee et al. reported a high-affinity,α-bungarotoxin (α-Bgtx)-sensitive receptor which was one of the first hints of a possible heteromeric α7* nAChR based on differential α-Bgtx sensitivity [21]. Soon, the functional evidence for heteromeric α7 receptors was reported in embryonic chick sympathetic neurons [22] and in intracardiac ganglion neurons [23]. Collectively, these pioneering works suggest that chick peripheral neurons likely contain heteromeric α7* nAChRs. Initial gene expression studies demonstrated that nAChR α7 and β2 subunits partially overlap on the cell somata of cultured hippocampal neurons [24]. Interestingly, Azam et al. reported that several cholinergic circuit brain regions predominantly expressed α7 and β2 subunit mRNAs, rather than α4 mRNA, and that these α7 and β2 subunit mRNAs are usually co-expressed [14], suggesting the possibility of α7β2 nAChRs in cholinergically-relevant neurons of the mammalian brain. We transfected α7 and β2 subunit mRNA into Xenopus oocytes and formed functionalα7-containingnAChRs (α7*nAChR)with different current kinetics and pharmacology than that of oocytes transfected with onlyα7 subunits [25]. In addition, we showed co-immunoprecipitation of α7 and β2 subunits from cotransfected TSA201 cells, further suggesting the co-assembly of α7β2 nAChRs. Subsequently, we reported findings consistent with a novel, naturally-occurring α7* nAChR subtype in rodent basal forebrain cholinergic neurons [26]. In these cells,α7 subunits were found to be co-expressed, colocalized and co-assembled with β2 subunit(s), as demonstrated by RT-PCR, immunohistochemistry, α7 and β2 subunit co-immunoprecipitation, and patch-clamp recordings (combined with pharmacological tools) using wild-type (WT) and nAChR β2 knockout (KO) mice [26]. We and coworkers further confirmed this heteromeric α7β2 pentamer using Förster resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF) microscopy in cells of the human epithelial cell line, SH-EP1, transfected with human α7 and β2 subunits [27]. Recently, pharmacological characteristics of expressed α7β2 nAChRs have been further profiled in Xenopus oocytes, demonstrating possibly different pharmacological properties compared to homomericα7 nAChRs [15], although differences are frequently subtle for some compounds tested to date. In addition to rodents,α7β2 nAChRs have also been found in human basal forebrain neurons [15] and cerebral cortical neurons [16]. Importantly, by using concatemeric technology, the α7β2 nAChRs with different, completely defined stoichiometric combinations and subunit associations have been recently characterized [15]. Collectively, by using these experimental techniques (Box 3), accumulated evidence demonstrates thatnAChRα7 and β2 subunits can co-assemble to forma functional heteromericα7β2 nAChR in the CNS.

Box 3. Techniques Used To Study nAChR Assembly, Expression, and Function.

Patch-Clamp Recording Technique

The patch-clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channel activity and whole-cell currents in neurons. Owing to high sensitivity and the ability to perform rapid solution exchange, this technique is highly suitable for studying ligand-gated ion channel function, especially for α7 nAChR-mediated currents because of the rapid activation and desensitization of α7 nAChRs during ligand exposure.

Reverse Transcription PCR (RT-PCR)

RT-PCR is one variant of PCR which is commonly used in molecular biology to detect the level of RNA expression. RT-PCR can be used to qualitatively detect gene expression through the creation of cDNA from RNA transcripts. Although traditional PCR and the RT-PCR both produce multiple copies of specific amplified DNA sequences, the applications of the two techniques are fundamentally different. Traditional PCR is used to exponentially amplify target DNA sequences while RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using traditional PCR. In addition to the qualitative study of gene expression, RT-PCR can be utilized for quantification of RNA expression, in both relative and absolute terms, by incorporating quantitative (q)PCR into the technique. The combined technique, described as quantitative RT-PCR or real-time RT-PCR, is considered to be the most powerful, sensitive, and quantitative assay for the detection of RNA levels.

Immunoprecipitation (IP)

IP is the technique of precipitating a protein antigen out of solution using an antibody that specifically recognizes and binds to that particular protein. This process can be used to isolate and concentrate a specific protein from a sample containing a mixture of thousands of different proteins. IP uses a secondary antibody (often immobilized to a solid phase) to crosslink the target and primary antibody complexes, forming a large multimolecular complex that can be easily separated from the liquid phase. Immunoprecipitation of intact protein complexes (i.e., antigen together with any proteins or ligands that are bound to it) is termed co-immunoprecipitation (Co-IP). Co-IP works by selecting an antibody that specifically targets a known protein that is believed to be a member of a larger complex of proteins. By targeting this known member with an antibody it may become possible to pull the entire protein complex out of solution and thereby identify unknown members of the complex that interact with the target protein. Co-IP is a powerful technique frequently used by molecular biologists to study protein–protein interactions.

Fluorescence Resonance Energy Transfer (FRET)

The mechanism of FRET involves a donor chromophore, initially in its electronic excited state, which may transfer energy to an acceptor chromophore in nonradiative fashion through long-range dipole–dipole coupling. The efficiency of this energy transfer depends on the inverse 6th power of the distance between donor and acceptor, making FRET able to measure nanometer scale distance and the changes in distance. Measurements of FRET efficiency can be used to determine if two fluorophores are within a specific distance of each other. Such measurements are used as a research tool in fields including biology and chemistry.

Total Internal Reflection Fluorescence (TIRF) Microscopy

TIRF microscopy (TIRFM) employs a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed. Prism-, objective-, and lightguide-based TIRFM produce the evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the surface of a glass, silica, or plastic slide. The evanescent wave is generated only when the incident light is totally internally reflected at the surface/water or surface/specimen interface. The evanescent electromagnetic field decays exponentially away from the interface, and thus penetrates to a depth of only approximately 200 nm into the sample medium. Thus TIRFM enables selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm in thickness) of cells. TIRF can also be used to observe the fluorescence of a single molecule, making it an important tool of biophysics and quantitative biology.

Receptor Concatemer Technology

nAChRs are natively assembled from individual subunits to produce functional pentameric assemblies. In native expression systems, a combination of intrinsic subunit properties and cellular chaperones are used to control subunit assembly. However, even under these circumstances, multiple receptor subtypes and/or stoichiometries can result. In heterologous expression systems the expression levels can be very different from those in native systems, and elements of the chaperoning system can be different or absent. Concatenated protein constructs encode multiple subunits (in this case of nAChRs) joined with short peptide linkers. In the case of fully concatenated pentameric nAChR constructs, this approach has been used to completely define subunit stoichiometry and subunit–subunit associations of nAChRs. This provides significant advantages in studying pure populations of desired nAChR subtypes, in probing the effects of particular subunit compositions/assembly orders, and in performing detailed structure–function studies.

Functional Properties of Heteromeric α7β2 nAChRs

The major functional feature of α7β2 nAChRs is their slower (relative to homomeric α7 receptors) whole-cell current decay kinetics (Figure 2), which has been confirmed in heterologously transfected Xenopus oocytes [16,25,26,28], natively expressed mouse basal forebrain cholinergic neurons [26] and hippocampal interneurons [29], and functional α7β2 nAChR concatemers [15]. Another functional property of α7β2 nAChRs is their lower whole-cell current amplitude (relative to homomeric α7 receptors) to nAChR agonists, which has been observed both in heterologously expressed systems and natively expressed neurons. Consistent with these properties, the single-channel conductance of α7-containing nAChRs is lower in hippocampal interneurons than in expressed homomeric α7 nAChRs [30]. However, cell-surface expression levels may also be an important contributor to the magnitude of the macroscopic currents observed. For example, concatemeric α7β2 nAChRs expressed in Xenopus oocytes have larger agonist-induced current amplitudes compared to those measured from homomeric α7 nAChR concatemers [15]. The insertion of one β2 subunit into the α7 concatemeric pentamer (at position 3) results in a threefold increase in current amplitude (compared to the homomeric α7 nAChR concatemer), and insertion of two β2 subunits into the α7 pentamer (at positions 2 and 4) results in a twofold increase in current amplitude [15]. These increases in macroscopic current seem likely to be due to increased surface expression of concatemeric constructs containing the β2 subunit. Co-expression of the α7 and β2 subunits does not result in any significant change in the current–voltage curve [25]. In addition to the altered channel decay kinetics and amplitude between heteromeric α7β2 nAChRs and homomeric α7 nAChRs in the CNS, it will be interesting to test other receptor/channel functions in the α7β2 nAChRs such as single-channel properties and Ca2+ permeability in the future.

Figure 2.

Figure 2

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Comparison of Current Kinetics Between α7 and α7β2 nAChR-Mediated Currents. (A) Choline-induced inward currents in dissociated rat ventral tegmental areas (VTA) dopamine (DA) neuron (left) and vertical diagonal band of Broca (VDB) cholinergic (Cho) neuron (middle) demonstrate a slow current kinetic and wide current duration in VDB cholinergic neurons. (B) In transfected Xenopus oocytes, compared to homomeric α7 nAChRs, the α7β2(1:1) nAChR-mediated current shows small amplitude, a slow decay constant, and longer current duration.

Pharmacological Properties of Heteromeric α7β2 nAChRs

One of the more intriguing questions is whether the heteromeric α7β2 nAChR exhibits an altered pharmacological profile compared to the homomeric α7 nAChR, including affinity and potency of nAChR agonists, antagonists, or allosteric modulators. Currently the accumulated data are still incomplete. Overall, often small changes in EC50 and IC50 values have been reported for multiple agonists and antagonists. However, these differences have often been inconsistent between reports examining the same compounds. This may reflect the difficulty of measuring subtle changes in pharmacological parameters, inconsistent subunit assembly order and/or stoichiometries between the various systems used, or a combination of both.

Agonists

Initial work demonstrates that the co-expression of the α7 and β2 subunits in Xenopus oocytes results in significant changes in pharmacology compared to expressed homomeric α7 nAChRs. For example, whereas carbachol and choline are full or near-full agonists for the homomeric α7 nAChR, they are only partial agonists for heteromeric α7β2 nAChRs [25]. In addition, the EC50 values for acetylcholine (ACh), carbachol, and choline significantly increased when the β2 subunit was co-expressed with the α7 subunit, suggesting lower receptor potency to nAChR agonists of the α7β2 nAChR compared to the homomeric α7 nAChR. This feature of heteromeric α7β2 nAChRs has been confirmed by several groups [15,27,28]. Impressively, Zwart et al. find that, except for ACh and epibatidine, which do not distinguish between the α7β2 and the α7 nAChR, α7β2 nAChRs display lower efficacy for 13 nAChR agonists tested, including three non-selective agonists (nicotine, cytisine, varenicline) and 10 selective α7 agonists (choline, carbamylcholine, DMAD, compound A, PNU-282978, SSR-108911, TC-1698, TC-7020, TC-5619, and EVP-6124) [28] (Table 1). In addition, they found that three selective α4β2 nAChR agonists (ABT-089, sazetidine A, ispronicline) fail to activate either the α7 or α7β2 nAChRs [28]. These pharmacological features of α7β2 nAChRs suggest that, while incorporation of β2 subunits into the α7 subunit pentamer formed functional heteromeric α7β2 nAChRs with altered function (compared to the homomeric α7 receptor), this does not shift the α7 nAChR to a ‘β2-like’ pharmacological profile. However, one of the most interesting findings is that some α7 partial agonists (e.g., compound A and DMBA) demonstrate significantly lower potency to activate α7β2 nAChRs compared to α7 nAChRs, and were therefore functional antagonists of α7β2 nAChRs. Therefore, compound A and DMBA may provide useful pharmacological tools to distinguish functional α7β2 nAChRs from α7 nAChRs [28].

Table 1.

Pharmacological comparison of nAChR agonists between α7 and α7β2 (1:10) expressed in Xenopus oocytes

Agonists α7 α7β2 (1:10) Reference
EC50 (μM) Emax (%) Hill EC50 (μM) Emax (%) Hill
ACh 180.0 113 1.4 300.0 (1.7) 115 (1.0) 1.6 #21
113.0 106 1.2 170.0 (1.5) 107 (1.0) 1.7 #16
Nicotine 36.0 87 2.2 123.0 (3.4) 45 (0.5) 1.5 #21
Choline 3100.0 116 1.7 5200.0 (1.7) 37 (0.3) 1.1 #21
972.0 108 2.1 1355.0 (1.4) 75 (0.7) 1.7 #16
CCh 800.0 90 1.4 1800.0 (2.3) 42 (0.5) 1.3 #21
779.0 118 1.6 923.0 (1.2) 76 (0.6) 1.4 #16
Epibatidine 1.3 101 1.3 1.4 (1.0) 91 (0.9) 1.4 #21
0.5 98 2.7 2.6 (5.2) 99 (1.0) 1.0 #16
Cytisine 28.0 71 2.3 113.0 (4.0) 16 (0.2) 1.5 #21
PNU-282987 5.0 91 0.8 2.7 (0.5) 19 (0.2) 1.3 #21
TC-1698 1.4 100 0.6 11.0 (7.9) 32 (0.3) 0.7 #21
TC-5619 0.4 106 1.1 32.0 (80.0) 14 (0.1) 0.8 #21
TC-7020 0.9 111 0.8 50.0 (55.6) 34 (0.3) 1.2 #21
EVP-6124 1.2 80 0.9 32.0 (26.7) 14 (0.2) 0.8 #21
Compound-A 1.1 75 0.3 40.0 (36.4) 12 (0.2) 1.2 #16
Compound-B 0.3 88 1.1 9.8 (32.7) 65 (0.7) 0.5 #21
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Note: The numbers after EC50 and Emax in α7β2 (1:10) panel indicate the ratio of α7β2/α7β2.

Antagonists

Most published works have found that α7β2 and α7 nAChRs display similar antagonism by α7-selective nAChR antagonists (e.g., MLA and α-Bgtx), which suggests that the predominant binding site for these antagonists for α7β2 nAChRs are likely located at the α7–α7 interface. By contrast, Murray et al. found a reduction in the MLA IC50 value (from 0.27 for α7 nAChR to 0.13 μM for α7β2 nAChR) in oocytes expressing α7β2 (1:10) nAChRs [27]. For the non-α7 nAChR antagonist, DHβE, which does not block homomeric α7 nAChRs, the α7β2 receptors in rodent basal forebrain cholinergic neurons and hippocampal interneurons are sensitive to DHβE (while the homomeric α7 nAChRs expressed in midbrain neurons are not) [26,29]. For heterologously expressed α7β2 nAChRs in oocytes, DHβE inhibition occurred only at the low concentration range, with an IC50 value of 4.58 ± 0.4 μM and was well fitted by a single Hill equation with an nH value of 1.2 ± 0.1 [27]. However, recently two groups, using either individual or concatemerically expressed α7β2 nAChRs in oocytes, have shown no difference in DHβE inhibition between α7β2 and α7 nAChRs [15,28]. Furthermore, there was no difference in the inhibition to two other nAChR antagonists (mecamylamine and the snake venom α-toxin) among individual α7, concatemeric α7, and concatemeric α7β2 nAChRs as well [15,28]. Table 2 summarizes the effects of nAChR antagonists on homomeric α7 and heteromeric α7β2 nAChRs.

Table 2.

Pharmacological comparison of nAChR antagonists between α7 and α7β2 oocytes

Agonists α7 α7β2 Reference
IC50 (μM) Bottom (%) Hill IC50 (μM) Bottom (%) Hill
MLA (oocytes) 0.00038 0 2.2 0.00030 (0.8) 0 (0) 1.7 (1:10) #21
(oocytes) 0.00089 1.7 0.0013 (1.5) 1.6 (1:10) #16
(Native) 0.00019 0 1.4 (VTA) 0.0012 (0.6) 0 (0) 1.3 (VDB) #19
DHβE (oocytes) 9.7 0 1.9 5.5 (1.1) 0 (0) 1.2 (1:10) #21
(oocytes) 47 2.7 18 (0.4) 1.1 (1:10) #16
(Native) >10 80 1.0 (VTA) 0.18 0 (0) 0.8 (VDB) #19
DMAB (oocytes) 24 49 2.0 9 (0.4) 15 (0.3) 1.9 (1:10) #20
Compound-A (oocytes) n.d. n.d. n.d. 26 6 0.6 (1:10) #20
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The n.d. means no detectable inhibition was found with Compound-A on α7 receptors.

Positive Allosteric Modulators

Because as mentioned above it is likely that the activation of α7β2 nAChRs by agonists is predominantly by action at the α7–α7 interface, and that the α7–β2 interface seems to be different from the α4–β2 interface, the functional α7–β2 interface may be a target for α7-selective positive allosteric modulator binding sites. While Murray et al. (2012) initially reported that α7β2 and α7 nAChR-mediated currents were not differentially potentiated by the α7 nAChR positive allosteric modulator PNU-120596 [27], Thomsen et al. recently compared the effects of PNU-120596 on human subunit α7 and α7β2 nAChRs heterologously expressed in Xenopus oocytes; they also found similar potentiation of α7 and α7β2 nAChR-mediated currents by this allosteric potentiator [16]. Therefore these results suggest that the site of action for the positive allosteric modulator PNU-120596 is likely at the α7–α7 interface.

Distribution of Heteromeric α7β2 nAChR in the CNS

Initial gene expression observations laid the groundwork for our following studies that provoked the hypothesis of the heteromeric α7β2 nAChRs [14]. Combining patch-clamp electrophysiological and single-cell RT-PCR analysis, Yakel and colleagues reported a strong correlation between expression of the α7 and β2 subunits in individual rat hippocampal interneurons [31], and Azam et al. found co-expression of nAChR α7 and β2 subunit mRNA in most cholinergic-associated brain areas including forebrain cholinergic neurons [14]. Following these reports, we recognized that rodent basal forebrain cholinergic neurons express heteromeric α7β2 nAChRs [26]. Later, we further confirmed that young mouse hippocampal interneurons also express functional α7β2 nAChRs [29], which is consistent with previous reports that the α7-containing nAChRs in rat hippocampal interneurons desensitized more slowly, and had a lower single-channel conductance, than homomeric α7 nAChRs [30,31]. However, Moretti et al. used α-Bgtx affinity purification and immunoprecipitation with anti-α7 subunit antibodies to isolate nAChRs containing α7 subunits from WT, α7 KO, or β2 KO mouse brain samples; they demonstrated that the α7β2 nAChRs were not detected in hippocampal tissues, but were detected in the forebrain, based on Western blot analysis of isolates using β2 subunit–specific antibodies [15]. Recently, Thomsen et al. reported the presence of heteromeric α7β2 nAChRs in human cerebral cortex [16]. These differences between studies may be due to genuine species differences or be a reflection of assay sensitivity because α7β2 nAChRs constitute a small portion of the overall α7 subunit-containing nAChR population in all regions examined so far. However, because cholinergic forebrain neurons are few in number, it is likely that even the smaller population of α7β2 nAChRs could be functionally relevant if they are located predominantly on only a small subset of cholinergic neurons (which project extensively to the rest of the brain). Using mouse cortical synaptosomes, Mehta et al. found that Aβ- and nicotine-induced Ca2+ responses appeared to largely involve α7* nAChRs, but the results using Aβ also indicated the possible presence of β2-containing receptors. In particular, by combining the use of the selective α7 nAChR antagonist, α-BgTx, with synaptosomes prepared from cortices of β2 subunit KO mutants, the α7* nAChR-coupled responses from these mice were smaller in amplitude when exposed to nM doses of Aβ, similar to what was observed for pM doses of Aβ applied to control cortical preparations. This result suggests the possibility of α7β2* nAChRs located in presynaptic terminals of cortical neurons [32]. In addition, Lykhmus et al. used a wide spectrum of nAChR subunit-specific antibodies, combined with nAChR α7 or β2 KO mice, to demonstrate the presence of α7β2 nAChRs in intracellular organelles (e.g., brain mitochondria) [33]. Although these α7β2 nAChRs may not function as classical ion channels, at least for regulating mitochondrial pore formation, activating these α7 nAChRs should affect intra-mitochondrial kinases involved in MPTP formation [34], suggesting that these mitochondrial α7β2 nAChRs play an important role in maintaining mitochondria resistance to apoptogenic factors. Taken together, the heteromeric α7β2 nAChRs have been identified in several brain regions, in pre- and postsynaptic compartments, as well as intracellular organelles such as mitochondria, where they may play an important role in mediating cholinergic modulations in brain function and diseases.

Assembly of Heteromeric α7β2 nAChRs

After the identification of functional native α7β2 nAChRs, the stoichiometry of this heteromeric nAChR was investigated in heterologous expression systems. One way to reveal the relationship of receptor stoichiometry and function is to express α7 and β2 nAChR subunit mRNAs at different ratios. In early work, Khiroug et al. compared agonist affinity (i.e., EC50 values) of α7β2 nAChRs with α7 and β2 subunit injection ratios of 1:1 and 1:3, and found a doubled EC50 value when the β2 subunit cDNA was increased; this suggested that the increased presence of the β2 subunit ratio results in a decrease in affinity of the α7β2 nAChR [25]. Furthermore, Zwart et al. compared receptor function between homomericα7 and heteromericα7β2 nAChRs by nuclear injections of cDNAs in 1:3 and 1:10 ratios, and found a significant reduction of current amplitude and slowed current desensitization kinetics for the α7β2 receptors, but with no difference in agonist affinity [28]. Both of these observations indicate that expression of functional α7β2 nAChRs is possible with multiple subunit stoichiometries. Recently, the use of concatemeric technology to study the stoichiometry of α7β2 nAChRs heterologously expressed in Xenopus oocytes has provided new insights, including direct confirmation of multiple functional α7β2 nAChR stoichiometries. For example, Moretti et al. constructed the fully pentameric α7* nAChR concatemers from human nAChR subunit sequences, and compared the function and pharmacological profiles of these α7* nAChR concatemers (both homomeric α7 and heteromeric α7β2) with the subunits arranged in the order α7–α7–α7–α7–α7 [(α7)5-nAChR homopentamer)], α7–α7–β2–α7–α7 [(α7)4(β2)1-nAChR], or α7–β2–α7–β2–α7 [(α7)3(β2)2-nAChR]. Each of the three concatemeric structures functionally expressed and exhibited ACh-induced currents. Compared to the homomeric α7 nAChRs from individual (i.e., unlinked) pentameric human α7 subunits, the concatemeric constructs had smaller current amplitudes, but both concatemeric heteromeric receptors [(α7)4(β2)1 and (α7)3(β2)2 nAChRs] had larger current amplitudes and slower decay kinetics (τslow) [15]. Pharmacologically, all three concatemeric constructs exhibited similar responses to different nAChR agonists; however, nicotine had significantly lower efficacy (normalized to that of ACh) at (α7)4(β2)1 and (α7)3(β2)2 α7β2 nAChR concatemeric structures compared to (α7)5-nAChR concatemer or unlinked homomeric α7 nAChRs [15]. In addition, the antagonist pharmacology of α7 (MLA) or β2 (DHβE) nAChRs were compared and found to be indistinguishable between the different α7 nAChRs [15]. Collectively, these concatemeric constructs provided new insights and improved understanding of the possible diverse stoichiometry of α7β2 nAChRs, and these data confirm some functional and pharmacological characteristics of α7β2 nAChRs previously reported in heterologous expression systems and native receptors in neurons. However, it should be noted that, while the subunit ratios and associations within concatemeric α7β2 nAChRs are completely known, the same is not true of either natively expressed α7β2 nAChRs or those assembled in artificial expression systems from unlinked subunits. It will be interesting and informative to further compare the function and pharmacology across receptors assembled in different ways.

Significance of the Heteromeric α7β2 nAChR

Because homomeric α7 nAChRs are predominantly expressed throughout the CNS and nonneuronal system, we considered the possible significance of heteromeric α7β2 nAChRs, and how might they function in vivo differently from homomeric α7 nAChRs.

Heteromeric α7β2 nAChR Is a Sensitive Target for Amyloid β Peptides

Because native α7β2 nAChRs were initially found in basal forebrain cholinergic neurons [26], this receptor may play an important role in the modulation of cholinergic signaling, and consequently brain cognitive function and disease pathology, such as in Alzheimer’s disease (AD). Consistent with this, we initially found that the α7β2 nAChRs in rodent basal forebrain cholinergic neurons exhibited high sensitivity to pathologically-relevant concentrations (e.g., 1 nM) of amyloid β peptide (e.g., oligomeric Aβ1–42) [26], as compared to homomeric α7 nAChRs where much higher concentrations of Aβ1–42 (e.g., 100 nM oligomeric) were needed for inhibition [35]. This finding was further confirmed by the sensitivity of heterologously expressed α7β2 and α7 nAChRs in Xenopus oocytes; heteromeric α7β2 nAChRs were much more sensitive to Aβ1–42 inhibition than homomeric α7-nAChRs [29]. Because the heteromeric α7β2 nAChR in basal forebrain cholinergic neurons is a sensitive target for pathologically-relevant levels of Aβ1–42 [36], this receptor may participate in the pathogenic process of AD. The mechanism that pathologically- relevant levels of Aβ1–42 use to inhibit α7β2 nAChRs in AD pathogenesis is still unclear, but there are at least two possibilities. First, the acute inhibition of α7β2 nAChRs by Aβ1–42 may directly lead to basal forebrain cholinergic signaling impairments in the hippocampus and subsequent learning and memory deficits [29]. Second, the Aβ1–42-sensitive α7β2 nAChRs may mediate chronic upregulation of α7-containing nAChRs because acute inhibition of α7 nAChRs triggers receptor upregulation [37–39]. In primary hippocampal neuronal cultures, we found that chronic treatment of neurons with Aβ1–42 (100 nM, fibrils or oligomers) upregulated α7 nAChR surface expression and function, which can be abolished by α7 nAChR antagonists and was absent in cultures prepared from α7 nAChR KO [38]. This upregulation by Aβ1–42 required a higher concentration (i.e., 100 nM) than we found was necessary to block the heteromeric α7β2 receptors, suggesting that the receptors responsible for this upregulation were homomeric α7 nAChRs [38]. Moreover, using human neuroblastoma (SH-SY5Y) cells differentiated by the addition of 10 μM retinoic acid to each dish, and incubating at 37 °C for 4 days to have cholinergic characteristics, we tested the effects of large Aβ1–42 aggregates (100 nM) on α7 nAChR function and cell toxicity, and found that chronic exposure of Aβ1–42 aggregates upregulated α7 nAChR function, and consequently resulted in cellular toxicity, which was prevented by pharmacological block of α7 (but not α4β2) nAChRs [39]. These results suggest that α7 nAChRs (likely homomeric) play an important role in Aβ1–42-induced mediation of neuronal excitability as well as cellular toxicity. It will be interesting to examine the effects of lower doses of Aβ exposure (e.g., ≤10 nM) on α7* nAChR expression and function in basal forebrain cholinergic neurons, where the heteromeric α7β2 nAChRs are expressed.

Heteromeric α7β2 nAChR Is a Sensitive Target for Volatile Anesthetic Agents

Neuronal nAChRs are important targets for anesthetic agents, in particular the heteromeric α4β2 nAChR subtype; the homomeric α7 nAChRs are less sensitive to general anesthetic agents [40,41]. The mechanism of this differential sensitivity to anesthetic agents between α4β2 and α7 nAChR is unclear. In previous studies using molecular dynamics simulations, Tang’s group found that, although multiple anesthetic binding sites were observed in α7, α4 and β2 subunits, anesthetic binding to a site at the interface between extracellular and transmembrane domains of the β2 subunit produced a profound change in protein dynamics that was likely to affect channel function [42–45]. Based on these results, they proposed a hypothesis to explain the lower sensitivity of α7 nAChRs to general anesthetics, in which the susceptibility to anesthetic perturbation in the β2 (but not α7) subunit underlies the functional sensitivity of α4β2 nAChRs to volatile anesthetics [42]. To test this hypothesis, we examined the effects of isoflurane on native α7β2 nAChRs expressed in rat basal forebrain cholinergic neurons and heterologously expressed α7β2 nAChRs in Xenopus oocytes, and found that isoflurane significantly inhibited native α7β2 nAChRs on basal forebrain cholinergic neurons but did not inhibit native α7 nAChRs on ventral tegmental area (VTA) dopamine neurons [46]. Similar results were also observed in heterologously transfected α7β2 and α7 nAChRs in Xenopus oocytes [46]. These results clearly suggest that, compared to homomeric α7 nAChRs, the heteromeric α7β2 nAChR is sensitive to anesthetic agents and therefore may play a role in the mediation of anesthetic effects in the CNS.

Inhibition of Heteromeric α7β2 nAChRs by α7 nAChR Partial Agonists

As mentioned above, some α7 nAChR partial agonists (e.g., compound A and DMBA) may demonstrate significantly lower efficacy for α7β2 nAChRs as compared to α7 nAChRs, and were therefore essentially functional antagonists for α7β2 nAChRs (rather than partial agonists for α7 nAChRs), providing useful pharmacological tools to clearly distinguish functional α7β2 from α7 nAChRs [28]. These findings are potentially highly significant because with such compounds one can selectively inhibit α7β2 nAChRs, and this will provide a novel experimental approach to evaluate the impact of α7β2 nAChRs in cholinergic signaling-modulated neural functions and disorders. However, given earlier-stated caveats concerning discrepancies in pharmacological parameters measured across multiple studies, perhaps reflecting differences in α7β2 nAChR assembly across the experimental models used, it will be important to confirm these exciting findings.

Concluding Remarks

The α7 nAChR is one of most highly expressed nAChRs in the CNS and mediates crucial cholinergic signaling in a variety of brain functions and disorders. The α7 nAChR was classically considered to be a homomeric nAChR. However, this idea has been recently challenged by accumulating evidence that demonstrates that a heteromeric α7 nAChR can be co-assembled and functional when formed with the β2 subunit in both heterologous expression systems and naturally in brain neurons. This novel type of α7β2 nAChR exhibits distinctive pharmacology and function from traditional homomeric α7 nAChRs, suggesting that it may play an important and unique role in the physiology, pharmacology, and pathophysiology in the CNS. However, there are several important questions about this novel type of α7β2 nAChR that need to be addressed in future studies (see Outstanding Questions). For example, while some small (and sometimes inconsistent) pharmacological differences have been seen, agonists that are highly selective for α7β2 nAChRs have yet to be discovered. Thus far, all α7 and α4β2 nAChR-selective agonists have not shown significant selectivity between heteromeric α7β2 and homomeric α7 nAChR nAChRs. This has been used to hypothesize that these agonists predominantly activate α7* nAChRs at the α7–α7 subunit interface [15,27]. If this is the case, what role in ligand recognition (if there is any) may be played by the α7–β2 interface? Is it possible that some ligands yet to be discovered do in fact act at the α7–β2 interface to alter α7β2 nAChR function and pharmacology? Second, what is the stoichiometry of native α7β2-nAChRs? Although the use of concatemers expressed in Xenopus oocytes provided insights into the understanding of α7β2 nAChR stoichiometry, these completely defined assemblies are subtly different functionally and pharmacologically from native α7β2 nAChRs in basal forebrain cholinergic neurons and hippocampal interneurons. Because fully concatemeric pentameric constructs faithfully replicate the properties of other nAChR subtypes (see for example [47–51]), this strongly suggests that concatemers with different ratios and assembly orders of α7 and β2 subunits need to be investigated, and that rigorous control of subunit ratios in unlinked heterologous expression systems is needed. Third, are functional α7β2 nAChRs expressed in other brain regions? Presently it has been shown that native α7β2 nAChRs are expressed in rodent basal forebrain cholinergic neurons and hippocampal interneurons, and in human cerebral cortical neurons, but not in human cerebellar and hippocampal neurons (at least at levels detectable with the techniques used to date). Fourth, what if any role do these heteromeric α7* nAChRs play during development and in the pathogenesis of disease? For example, the fact that α7β2 nAChRs are sensitive to pathologically-relevant levels of Aβ1–42 suggests that these receptors may have a role in the pathogenesis of AD. Furthermore the development of selective α7β2 nAChR ligands may help to address the role of these receptors in AD and other disorders and diseases linked to the α7 receptor (e.g., Parkinson’s disease and schizophrenia) [52–55]. Fifth, the differences in Ca2+ permeability between α7 homomeric and α7β2 heteromeric receptors have not been investigated. Even in the absence of a change in relative Ca2+ permeability, the significantly slower current kinetics might mean that significantly more overall Ca2+ influx will occur per stimulation. This could be an important difference with regard to the functional impact of the α7β2 nAChRs. Sixth, are there other differences in functional properties (e.g., single-channel properties, conductance, and open probability) between α7β2 and α7 nAChRs? This information will tell us more about the potential role of the α7–β2 interface and the influence of β2 subunits on the properties of the α7β2 nAChRs. Clearly, we are at an early stage in our attempts to parse the contribution of α7β2 nAChRs in cholinergic modulation in brain function and disorders. Fortunately, the recent application of experimental tools (e.g., concatemeric technology, FRET) and early indications of (albeit somewhat incompletely/inconsistently characterized) novel α7β2 nAChR selective compounds (e.g., amyloid-β, compound A, DHβE, and DMBA) are hopeful signs. It seems likely that further developments in these regards will drive new investigations designed to address these important questions in ever more rigorous and resolving ways. Finally, in addition to heteromeric α7β2 nAChRs, other possible combinations of α or β subunits with the α7 subunit may form other types of heteromeric receptors, such as α7β4 or α7β3 nAChRs. It may even be possible that α7 subunit can assemble with α4β2 nAChRs to form a more complex α7α4β2 nAChR with altered α4β2 receptor function and pharmacology. All these possibilities should be tested in future studies.

Outstanding Questions.

What role in ligand recognition (if there is any) may then be played by the α7–β2 interface? Is it possible that some ligands yet to be discovered do in fact act at the α7–β2 interface to alter α7β2 nAChR function and pharmacology?

What is the stoichiometry of nativeα7β2 nAChRs? Although the use of concatemers expressed in Xenopus oocytes provided insights into the understanding of α7β2 nAChR stoichiometry, these completely defined assemblies are subtly different functionally and pharmacologically from native α7β2 nAChRs in basal forebrain cholinergic neurons and hippocampal interneurons.

Are functionalα7β2 nAChRs expressed in other brain regions? Presently it has been shown that native α7β2 nAChRs are expressed in rodent basal forebrain cholinergic neurons and hippocampal interneurons, as well as in human basal forebrain and cerebral cortical neurons.

What if any role do these heteromeric α7* nAChRs play during development and in the pathogenesis of disease? For example, the fact that α7β2 nAChRs are sensitive to pathologically- relevant levels of Aβ1–42 suggests that these receptors may have a role in the pathogenesis of AD. Furthermore, the development of selective α7β2 nAChR ligands may help to address the role of these receptors in AD and other disorders and diseases linked to the α7 receptor.

Are there other differences in functional properties (e.g., single-channel properties, conductance, and open probability) between α7β2 and α7 nAChRs? This information will tell us more about the potential role of the α7–β2 interface and the influence of β2 subunits on the properties of the α7β2 nAChRs.

Are there differences in Ca2+ permeability between α7 homomeric and α7β2 heteromeric receptors? Even in the case of absence of a change in relative Ca2+ permeability, the significantly slower current kinetics might mean that significantly more overall Ca2+ influx will occur per stimulation. This could be an important difference with regard to the functional impact of the α7β2 nAChRs.

Trends.

There is a novel type of heteromeric α7β2 nAChR that is expressed in heterologous expression systems and naturally in brain neurons.

Compared to classic homomeric α7 nAChRs, the α7β2 nAChR exhibits distinctive function and pharmacology.

Native α7β2 nAChRs are specifically expressed in brain regions associated with learning andmemory, such as basal forebrain and hippocampus, suggesting an important target for cholinergic modulation of brain cognitive function.

The α7β2 nAChRs are highly sensitive to pathologically-relevant levels of amyloid β peptides, suggesting a new target for Alzheimer’s disease pathogenesis and therapeutics.

Acknowledgments

This work was supported by grants from the Barrow Neuroscience Foundation to J.W. and P.W., Arizona Alzheimer Disease Consortium to J.W. and Q.L., NIH (R01GM66358 and R01GM56257) to P.T., NIH (R21DA026627) to P.W., the Intramural Research Program, NIEHS/NIH to J.L.Y., the Innovation Fund in Denmark, COGNITO to J.D.M., and National Natural Science Foundation of China (No. 31171089) to J.X.S.

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