Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Acetylcholine (ACh) binds to two major subclasses of cholinergic receptors in the central nervous system (CNS): the muscarinic and neuronal nicotinic ACh receptors (nAChRs), which mediate not only between-neuron communications but also the long-lasting modifications that occur during development. ACh acts on muscarinic ACh receptors to regulate cell proliferation and on nAChRs to regulate neurite outgrowth and pathfinding by neuronal growth cones (reviewed in Role and Berg, 1996; Zoli, 2000).

nAChRs are cationic channels whose opening is controlled by ACh. They are mainly involved in fast synaptic transmission in the autonomic nervous system (Berg et al., 2000), but also have regulatory functions in the CNS. Brain nAChRs are predominantly localized at presynaptic sites, where they influence the activity of various neurotransmission systems by regulating the release of specific neurotransmitters, such as ACh, dopamine, norepinephrine, serotonin, -aminobutyric acid, and glutamate (reviewed in Wonnacott, 1997; MacDermott et al., 1999).

nAChRs include a variety of subtypes, a heterogeneity that is attributable mainly to the diversity of the genes encoding the receptor subunits. Twelve vertebrate genes coding for nAChR subunits have so far been cloned (2-10 and 2-4), and a number of subtypes with different pharmacological and functional properties can be generated from the homopentameric or heteropentameric assembly of these subunits in heterologous systems. The homomeric channels can be obtained by the expression of 7, 8, or 9 subunits, whereas the heteromeric channels come from the coexpression of different combinations of 2, 3, 4, or 6 and 2 or 4 in presence or absence of 5 or 3 subunits (reviewed in McGehee and Role, 1995; Gotti et al.1997; Clementi et al., 2000; Lindstrom, 2000).

The pharmacological and functional properties of nAChR subtypes are mainly determined by the pentameric arrangements of their subunits, although post-translational events, transport to different membrane regions, and/or binding to linker proteins can also affect their function. The fact that more than one type of  or  subunit can coassemble in a single pentameric receptor greatly increases the number of possible receptor subtypes present in the nervous system, but not all of these potential subtypes are actually expressed because some still unknown mechanisms prevent the formation of some possible subunit combinations (reviewed in Lindstrom, 2000).

Given that the effects of ACh on neuronal development and functions after the establishment of synaptic contacts depend on the nAChR subtype expressed at each stage, it is very important to identify and investigate the properties of the subtypes expressed in the nervous systems.

7 and 42 are the predominant subtypes expressed in vertebrate brain, whereas the 34 subtype predominates in the autonomic nervous system (Gotti et al., 1997; Lindstrom, 2000). However, subtypes containing the 2, 5, 6, 3, and 4 subunits can be found in more limited CNS regions (Forsayeth and Kobrin, 1997; Lindstrom, 2000). The presence of these minor subtypes is also suggested by studies on knock-out animals: functional and ligand binding studies of animals lacking the 2 subunit suggest that 4-containing receptors are also present in restricted areas associated with an 3, 2, or 4 subunit (Picciotto et al., 1995; Zoli et al., 1998).

Because selective ligands for specific nAChR isoforms are still scarce, our group has devised an alternative approach toward identifying and characterizing the native subtypes present in the chick nervous system by preparing a series of antibodies (Abs) that specifically recognize all of the known subunits. Using this approach, we have recently been able to identify several new subtypes: 6- and 3-containing receptors in retina (Vailati et al., 1999, 2000), and the 252 subtype in chick optic lobe (Balestra et al., 2000).

In this study, we used a sequential immunodepletion procedure to identify the presence of an 44 subtype in chick retina, and then characterized its subunit composition and pharmacological profile and compared it with that of the transfected 44 subtype.

Furthermore, to study the relative contribution of the  or  subunits to the pharmacological profiles of the subtypes, we compared the pharmacological properties of the transfected chick 44 and 64 subtypes.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Antibody Production and Characterization. The polyclonal Abs against the 2, 3, 4, 5, 6 7, 8, 2, 3, and 4 chick peptides were raised and characterized as described by Vailati et al. (1999, 2000) and Balestra et al. (2000). For most of the subunits, two different peptides were chosen: one located in the cytoplasmic loop between M3 and M4 (CYT), and the other located at the COOH terminal (COOH). Each anti-peptide Ab was affinity purified by incubating the serum with an affinity resin made by coupling the corresponding peptide to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. The monoclonal Ab 299 raised against rat brain nAChR and directed against the 4 subunit (Whiting and Lindstrom, 1988) was purchased from RBI. The affinity-purified Abs were bound to CNBr-activated Sepharose at a concentration of 1 mg/ml, and the columns used for immunopurification.

Receptor Subtype Immunopurification

The 44 Subtype. The retina extracts were prepared as previously described by Vailati et al. (1999); every experiment involved the use of 150 g of chick eyes. The tissue was homogenized in an excess of 50 mM sodium phosphate, pH 7.4, 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM PMSF for 2 min in an ultraTurrax homogenizer. The homogenate was then diluted and centrifuged for 1.5 h at 60,000g.

Receptor Immobilization by Subunit-Specific Antibodies.

The affinity-purified anti-4 or anti-4 Abs were bound to microwells (Maxi-Sorp; Nunc, Wiesbaden, Germany) by means of overnight incubation at 4°C at a concentration of 10 µg/ml in 50 mM phosphate buffer, pH 7.5. On the following day, the wells were washed to remove the excess of unbound Abs, and then incubated overnight at 4°C with 200 µl of 2% Triton X-100 retina membrane extract containing 50 to 100 fmol of [³H]Epi binding sites, which was prepared by sequentially immunodepleting the extract with the anti-6, anti-3, and anti-2 Abs as described above. After incubation, the wells were washed and the presence of immobilized receptors revealed by means of [³H]Epi binding.

Immunoprecipitation of [³H]Epi-Labeled Receptors by Anti-Subunit Specific Abs during Retina Development.

The subunit content of the purified and transfected 44 subtypes was determined by immunoprecipitation using chick subunit-specific Abs as described previously (Vailati et al., 1999, 2000; Balestra et al., 2000).

The eyes and retinas were dissected from in ovo chicks on embryonic days 7, 11, 14, and 18 (E7, E11, E14, and E18) and from 1-day-old chicks (P1), immediately frozen in liquid nitrogen and stored at 80°C for later use. No differences in the binding properties of the fresh and frozen tissues were observed. For every experiment, the extracts of the tissues were prepared as described above, labeled with 2 nM [³H]Epi, and incubated overnight with a saturating concentration of affinity-purified IgG (20 to 30 µg). The immunoprecipitation was recovered by incubation of the samples with beads with bound goat anti-rabbit IgG (Tecnogenetics, Milau, Italy). The level of Ab immunoprecipitation was expressed as the percentage of [³H]Epi-labeled receptors immunoprecipitated by the indicated antibodies, taking the amount present in the Triton X-100 extract solution before immunoprecipitation as 100%.

Binding Assay and Pharmacological Experiments

(±)-[³H]Epi with a specific activity of 66.6 Ci/mmol was purchased from PerkinElmer Life Science Products (Boston, MA); nonradioactive Epi was from RBI/Sigma (Natick, MA). The drugs MG624 and F3 have been synthesized in our laboratory according to Gotti et al. (1998). -Conotoxin MII (MII) was a generous gift from M. M.; nonradioactive -bungarotoxin (Bgtx) and the drugs cytisine (Cyt), ACh, carbamylcholine (Carb), 1,1-dimethyl-4-phenylpiperazinium (DMPP), nicotine (Nic), methyllycaconitine (MLA), dihydro--erythroidine (DHE), MII, d-Tubocurarine (d-TC), hexamethonium (Hex), and decamethonium (Dec) were from Sigma.

Membrane. Binding to membrane homogenate obtained from BOSC 23 cells transfected with the 44 and 64 subunits were performed overnight by incubating aliquots of the membrane with [³H]Epi concentrations ranging from 0.005 to 5 nM at 4°C. Nonspecific binding (averaging 5 to 10% of total binding) was determined in parallel by means of incubation in the presence of 100 to 250 nM unlabeled Epi. A final concentration of 10 µg/ml of the protease inhibitors leupeptin, bestatin, pepstatin A, aprotinin, and 2 mM PMSF was added to the incubation mixture to block possible proteolysis during the long incubation time of the assays. At the end of the incubation, the samples were centrifuged and washed once with 10 sodium phosphate, pH 7.4, plus 50 mM NaCl, the pellet was dissolved with 2N NaOH, and the filters counted in a -counter.

[³H]Epibatidine Binding to Solubilized Receptor. Tissue extract binding was performed using DE52 ion-exchange resin (Whatman, Maidstone, UK) as previously described (Vailati et al., 1999). The binding techniques for immunoimobilized subtype as well as the data analysis were the same as those described previously (Vailati et al., 1999).

cDNA and Expression Vectors

The cDNAs encoding chick neuronal nAChR 4, 6, and 4 subunits cloned in the SV40-based expression vector Flip (Couturier et al., 1990; Nef et al., 1998) were kindly provided by Dr. Marc Ballivet (University of Geneva, Switzerland).

Expression of nACHR Subunits in BOSC23 Cells

Transient transfections of the nAChR subunits were carried out in the retroviral packaging cell line BOSC 23, as described previously (Ragozzino et al., 1997). The cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (Hyclone, Logan, UT). The subunit cDNAs were added in equivalent amounts (8 µg each per 100-mm dish). Between 8 and 12 h after transfection, the cells were washed twice and fed again with DME- containing 10% fetal calf serum. The cells were collected in ice-cold phosphate-buffered saline (Life Technologies) 36 to 48 h after transfection, and stored at 70°C.

Materials

The protease inhibitors, cholinergic ligands, Triton X-100, and anti-rabbit and anti-rat antisera were purchased from Sigma, the nonradioactive Epi from RBI/Sigma, CnBr-activated Sepharose 4BCL and ¹²⁵I-Protein A from Amersham Pharmacia Biotech, (±)[³H]Epi from PerkinElmer Life Sciences, and the reagents for gel electrophoresis from Bio-Rad Laboratories (Hercules, CA).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Epibatidine Binding Receptors in the Retina during Development. We have reported previously (Vailati et al., 1999) that there is a high level of expression of [³H]Epi-labeled receptors in 1-day-old chick retina. To investigate the developmental expression of these receptors, we performed binding studies using 2 nM [³H]Epi and 2% retina extracts obtained from chicks on E7, E11, E14, E18, and P1, and detected 50.3 ± 2, 162 ± 9, 220.7 ± 39, 264 ± 14, and 278 ± 21 fmol/mg of protein (mean ± S.E.M. of three experiments), respectively. If the binding to the same extracts was performed in the presence of 1 µM cold Bgtx, it decreased to 41.4 ± 2.9, 105.7 ± 7, 155 ± 19, 199.9 ± 21, and 204.3 ± 16 fmol/mg of protein, respectively (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Developmental changes in [3H]epibatidine-binding receptors expressed in the retina. The retinas were dissected from the embryos at the indicated times and frozen. Triton X-100 extracts (2%) were prepared from the tissues and assayed for [3H]Epi binding, which was performed in the absence () and presence () of 1 µM Bgtx. Data are mean values ± S.E.M. from three experiments performed in triplicate.

44 Subtype Identification. We have shown previously that the [³H]Epi binding receptors present in P1 chick retina are a heterogeneous population: the majority contain the 4 subunit, but there is a subpopulation that also contains the 2 subunit with or without the 4 subunit. Furthermore, they are also very heterogeneous in terms of their  subunit content (Vailati et al., 1999). Using anti-6 subunit-specific Abs, we immunodepleted the large majority of 6-containing receptors. The flow-through of the 6 affinity column still had receptors containing the 3, 2, and 4 subunits, and so we used anti-3 and anti-2 Abs in sequence, to immunodeplete the retina extract of the residual 3 (Vailati et al. 2000) and 2-containing receptors. Further immunoprecipitation of the flow-through obtained from the sequential columns confirmed the almost total depletion of receptors containing the 6, 3, and 2 subunits (see Table 1), and indicated the presence of 25% of ³H-labeled receptors that were immunoreactive to the 4 and 4 subunits.

View this table: [in this window] [in a new window]   TABLE 1 Percentage of immunoprecipitation of chick retina extracts labeled with [3H]Epi before and after immunodepletion with anti-6 -3 and -2 Abs by anti-subunit chick specific Abs. The immunoprecipitation was carried out as described under Experimental Procedures using saturating concentrations of anti-subunit Abs. The results are expressed as the percentages of [3H]Epi labeled receptors, taking the amount of receptor present in the solution before immunoprecipitation as 100%. The percentage of immunoprecipitation was subtracted from the value obtained in control samples containing an identical concentration of normal rabbit IgG. The values are the means ± S.E.M. of five determinations.

Subunit Composition of the 44 Subtype. The retina extract obtained after immunodepletion with the anti-6, -3, and -2 Abs was incubated with anti-4 Abs bound to Sepharose, and the bound receptors were eluted by competition with the 4 peptide or glycine pH 2.2.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Immunoprecipitation analysis of the subunit content of the native and transfected 44 subtypes. Top, the 44 retina subtype was purified as described under Experimental Procedures. After extensive dialysis to remove the 4 peptide used for the elution of the receptors from the affinity column, the receptors were labeled with 2 nM [3H]Epi and immunoprecipitated using saturating concentrations (20-30 µg) of anti-2-CYT, anti-3-CYT, anti-4-COOH, anti-4-CYT, anti 5-COOH, anti-6-CYT, anti-7-CYT, anti-8-CYT, anti-3-CYT, anti-4-COOH, and anti-4-CYT. Bottom, the BOSC23 cells were transiently transfected with chick 4 and 4 cDNA as described under Experimental Procedures. A 2% Triton X-100 extract was prepared from the cells and the receptors were labeled with 2 nM [3H]Epi and immunoprecipitated with the same Abs as in the top. The results are expressed as percentages of the [3H]Epi-labeled receptors, taking the amount of receptor present in the solution before immunoprecipitation as 100%. The percentage of immunoprecipitation was subtracted from the value obtained in the control samples containing an identical concentration of normal rabbit. Mean values ± S.E.M. of three determinations performed in triplicate.

View larger version (76K): [in this window] [in a new window]   Fig. 3.   Western blot analysis of the immunopurified 44 subtype. The receptors bound to the 4 Abs were eluted by incubation with 100 µM 4 peptide, concentrated, and separated on 9% acrylamide SDS gel, electrotransferred to nitrocellulose, and probed with the indicated anti-subunit specific Abs. The molecular mass markers (top to bottom) are 97 kDa, 67 kDa, 45 kDa, and 31 kDa.

Pharmacological Profile of the Native 44 Subtype and Comparison with the Transfected Subtype. The pharmacological experiments were all carried out on receptors immobilized by the corresponding anti-4-CYT specific Abs as described under Experimental Procedures. The 44 receptors bind [³H]Epi with high affinity; the K_d value calculated from 10 separate experiments was 11 pM (CV, 17%).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Saturation curve of [3H]Epi binding to immunoimmobilized native retina 44 receptors, and its Scatchard analysis (insert). The immunoimmobilized receptors were incubated overnight at 4°C with the indicated concentrations of [3H]Epi to measure total binding, and also in the presence of 100 nM Epi to measure aspecific binding. The total () and aspecific binding () shown is that obtained from a representative experiment; the Kd value of 11 pM [coefficient of variation (CV) = 17%] was calculated by simultaneously fitting 10 separate experiments. The Scatchard plot of the saturation curve shows the presence of a single class of high-affinity sites.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Inhibition by nicotinic agonists and antagonists of [3H]Epi binding to native immunoimmobilized retina 44 receptors. The receptors immunoimmobilized on the anti-4 CYT Abs (as described under Experimental Procedures) were preincubated for 30 min at 20°C with the indicated concentrations of nicotinic ligands; [3H]Epi was then added at a final concentration of 250 pM, and the mixture left overnight at 4°C. The curves were obtained by fitting three separate experiments using the LIGAND program (Munson and Rodbard, 1980). In each experiment, each dilution of the drug was tested in triplicate. All of the values are expressed in relation to [3H]Epi specific binding to the receptors (considered as 100%).

View this table: [in this window] [in a new window]   TABLE 2 Pharmacological characterization of native and transfected chick subtypes The Kd and Ki values were derived from the [3H]Epi saturation and competition binding curves to the immunoimmobilized native 44- and 64-containing subtypes and to homogenates of BOSC 23 cells transfected with the chick 44 and 64 subtypes. The curves obtained from three separate experiments were fitted using a nonlinear least squares analysis program and the F test according to Munson and Rodboard (1980). The numbers in brackets represent the percentage of CV.

Pharmacology of the Transfected 64 Chick Subtype. To study the role of the 4 and 4 subunits in the definition of the pharmacological profile of the subtype, we characterized the profile of the transfected chick 64 subtype (which has the same 4 subunit but a different  subunit) and compared it with that of the tranfected 44 subype.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   MII toxin inhibition of the [3H]Epi binding to native and transfected 4 containing subtypes. The native immunoimmobilized retina 64-containing () and 44 receptors (), as well as the cell homogenates of the human BOSC23 cells transfected with the 44 () or 64 (), were preincubated with the indicated concentration of MII toxin for 30 min; a final concentration of 250 pM [3H]Epi was added and left overnight. The results are expressed as in Fig. 5.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The pharmacological characteristics of chick, rat, and human 44 subtypes have been studied previously by electrophysiological and binding studies on receptors expressed in heterologous systems (Luetje and Patrick, 1991; Chavez-Noriega et al., 1997; Ragozzino et al., 1997; Stauderman et al., 1998), but this is the first biochemical and immunological demonstration of its presence in vertebrate CNS.

The results described here, together with those of our previous studies, indicate that 70 to 75% of the Bgtx-insensitive [³H]Epi-labeled receptors in chick retina contain the 4 subunit, which is the predominant retina subunit at P1. This subunit it is coassembled with the 6 subunit in 30 to 35% of the receptors (Vailati et al., 1999), with 3 in 5 to 10% of the receptors (Vailati, 2000), with 2 in 10 to 15%, and with 4 in 20 to 25%.

No direct evidence exists for the physiological role of these subtypes, but they might be involved in fine tuning the spontaneous activity required for the development of circuits in the retina and/or the formation of the appropriate retina-tectum connections. This role of nAChRs in the retina circuits is also suggested by the recent findings of altered spontaneous activity patterns in the retina of mice lacking the 2 and/or 4 nicotinic subunits (Bansal et al., 2000).

We have shown that both the Bgtx-sensitive and -insensitive receptors in the retinas of 1-day-old chicks contribute to the high affinity of [³H]Epi binding. The Bgtx-sensitive receptors are those that contain the 7 and/or 8 subunits, whereas the Bgtx-insensitive receptors include multiple subtypes, 25% of which contain the 4 and 4 subunits. These [³H]Epi-labeled receptors increase by 5- to 6-fold during retina development. At E7, most of the Bgtx-insensitive [³H]Epi binding is caused by receptors containing the 4, 3, and 2 subunits (S.V., M.M., and C.G., unpublished observations) whereas a large number of receptors also contain the 3, 4, and 6 subunits at P1. We (Gotti et al., 1994b) and others (Keyser et al., 1993) have previously shown that there is also a developmental increase in chick retina Bgtx binding receptors, which correlates with an increase in the number of receptors containing the 8 subunit.

Having established that P1 was the developmental time with the largest increase in [³H]Epi-labeled receptors insensitive to Bgtx, we used a series of immunodepletetion procedures to purify the native 44 subtype from the retina of 1-day-old chicks.

We analyzed the subunit composition of the purified 44 subtype by means of Western blot and immunoprecipitation experiments using Abs directed against all of the known chick nicotinic subunits. The blots of the purified subtypes were recognized only by the Abs directed against the 4 and 4 subunits. These results were confirmed in the immunoprecipitation studies in which only the anti-4 and 4 Abs immunoprecipitated more than 63% of the immunopurified [³H]Epi labeled receptors. To control the specificity of our immunoprecipitation studies, we performed the same immunoprecipitation experiment on 2% Triton extracts obtained from 44 transfected BOSC 23 cells. We obtained the same qualitative results with a higher recovery (more than 83% of the [³H]Epi-labeled receptors were immunoprecipitated), which suggests that the lower recovery of the purified receptors is probably caused by partial proteolysis during the long purification processes. The absence of immunoprecipitation with the other Abs in the native receptors is caused by the lack of subunits, because the same Abs were able to immunoprecipitate the receptors containing the corresponding subunits in control experiments.

Binding studies of the 44 subtype showed no difference in the affinity of the native and transfected subtypes for a number of nicotinic ligands: both had nanomolar affinity for the agonists and the DHE antagonist, and micromolar affinity for the toxins MII and MLA.

The highest agonist affinity was for Epi followed by Cyt, and the K_i values of ACh, Epi, and Cyt were very similar to those reported in the oocyte-transfected rat 44 subtype (Parker et al., 1998). The chick 44 subtype has a higher affinity for DhE than the native 42 subtype (Balestra et al., 2000), which is in agreement with the results obtained in electrophysiological experiments using oocyte-expressed rat (Harvey et al., 1996) and human subtypes (Chavez-Noriega et al., 1997).

Comparison of the pharmacological profile of the native 44 subtype with that of the 64 subtype also present in chick retina shows that the two subtypes have different K_i values for the toxin MII, DHE, d-TC, and F3. Because our purified 64-containing receptors make up a heterogeneous population in which 40 to 50% also have an additional 3 and/or 3 subunit, we investigated the affinity of these and other nicotinic ligands in the transfected 64 chick subtype. The pharmacological profile of the 64 subtype was similar but not identical to that reported previously for the native subtype: it has a high affinity for agonists, micromolar affinity for DHE, and an even higher affinity for the toxins MII (K_i = 4.5 nM) and MLA (K_i = 247 nM) and for the oxystilbene derivatives F3 (K_i = 264 nM) and MG624 (K_i = 440 nM). The high affinity for the MLA toxin is in agreement with the electrophysiological results obtained by Fucile et al. (1998), who found that 10 µM MLA is able to block the ACh-induced current in the same transfected subtype.

The pharmacological properties of the transfected 44 subtype reflect those of the native receptors, but the transfected and native 64 subtypes differ in terms of the absolute K_i values of some antagonists, thus suggesting that the presence of the 3 and/or 3 subunit may play a role in the definition of antagonist affinity in the native 64 subtype. The binding affinity of agonists and antagonists depends on both the  and  subunits (Parker et al., 1998). The greatest difference occurs as a consequence of changing the  subunit, but differences are also seen when the  subunit is changed. In the present study, we found that the K_i values of the MII toxin for the transfected 64 and 44 subtypes (which differ only in terms of the  subunit), are more than 400 times different. This result allows us to conclude that the MII toxin has a high affinity for the chick 64 subtype (K_i = 4.5 nM), and that this high affinity is mainly caused by the 6 subunit because both the native and transfected 44 subtypes have only micromolar affinity for MII.

The results obtained in binding studies are in agreement with the very recent finding by Kuryatov et al. (2000) that MII toxin not only inhibits the ACh-induced currents in the 32 oocyte-expressed subtype (as also reported previously by Cartier et al., 1996) but also potently inhibits both the chimeric 6/3 and 6/4 receptors containing either 2 or 4 subunits.

The high affinity of MII toxin on 6-containing receptors could be very important for dissecting the role of this subtype in brain function and, in particular, for improving our understanding of the addictive properties of nicotine. It has been suggested that the behavioral effects of nicotine depend on dopamine (Di Chiara, 2000), and mRNA for the 6 subunit is in dopaminergic nuclei projecting to the striatum (Le Novère et al., 1996) and MII toxin partially blocks the dopamine release from striatal synaptosomes (Kulak et al., 1997).

It is difficult to attribute specific functional roles to the 44 subtype in the CNS because its presence has only been demonstrated in chick retina and could be species-specific. Furthermore, studies performed in KO mice suggest that, if present, it is only a minor subtype: ligand binding and electrophysiological studies in ₂ KO animals (Zoli et al., 1998) have suggested that 44 receptors could be present in the interpeduncular nucleus and medial habenula, but the results of later binding studies of 4 KO animals (Marubio et al., 1999) make this possibility very unlikely.

It has recently been found that cocaine, a drug of abuse that primarily blocks the dopamine and serotonin transporters, also affects the heterologously expressed 44 rat subtype at concentrations compatible with those present in the serum of cocaine users (Francis et al., 2000). If this is proven true for the native subtype, a new pharmacological tool will be available for the study of this subtype in vivo.