Introduction

Summary Introduction Materials & Methods Results Discussion References

The family of nicotinic AChRs consists of subunits termed 1-9, 1-4, , , and . All of these subunits, except the "orphans" 6 and 3, have been shown to function as components of ACh-gated cation channels either as homomers or in combination with one or more other AChR subunits (1-4).

Despite the fact that the cDNA sequence of the 6 AChR subunits has been known for several years (1), little data are available regarding the properties of this subunit. These data are limited to mentions in reviews of cDNA sequences of rat and chick subunits (4, 5), a recently submitted cDNA sequence of the human subunit (6), and published abstracts concerning 6 mRNA distribution in rat brain and cochlea (7, 8). High levels of sequence homology between 6 and 3 AChR subunits (>75%) and other features common to all functional nicotinic subunits (5) indicated that 6 subunits should form functional AChRs serving as a ligand-binding subunit. However, difficulties in obtaining functional AChRs formed exclusively or partially by this subunit have suggested that 6 may serve a structural role in combination with other  and subunits as 5 does (9, 10), that it may require the presence of another subunit yet to be identified to function as an AChR, or that 6 may function as a receptor for some other ligand yet to be identified.

We show that 6 subunits can, in fact, act as ligand-binding subunits in functional AChRs. Here, we report cloning of cDNAs encoding 6 AChR subunits from a chicken cochlea library and a 4 AChR subunit from a human neuroblastoma SH-SY5Y library. When expressed in Xenopus laevis oocytes together with the human 4 subunit, chicken or rat 6 AChR subunits form nicotinic ligand-gated cation channels with novel pharmacological properties. Identification of this new subtype of AChR may prove important for understanding the pharmacological properties of centrally acting cholinergic ligands with possible therapeutic significance.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Isolation of chicken 6 and human 4 cDNA clones. A lambda Zap II cDNA library (~9 × 10⁶ plaques) using chick cochlear mRNA, constructed by Stratagene (La Jolla, CA), was kindly provided by Dr. Paul Fuchs (Johns Hopkins University, Baltimore, MD). The chicken 6 cDNA was obtained by screening ~5 × 10⁵ plaques from this library at low stringency using previously cloned chick 3, 4, 5, 7, 8, and 2, rat 2, 3, and 4, and human 1, 1, , and  full-length or nearly full-length cDNA probes. The human 4 cDNA clone was obtained by screening a previously described lambda Zap II cDNA library (11) constructed using mRNA isolated from the human neuroblastoma cell line SH-SY5Y with human 3, 4, 5, 7, 2, and 4 and rat 2, 3, and 6 full-length or nearly full-length cDNA probes. All rat AChR subunit cDNAs used were kindly provided by Drs. Stephen Heinemann and Jim Boulter (Salk Institute, San Diego, CA). A human 5 cDNA clone and a partial 4 cDNA clone were kindly provided by Dr. Francesco Clementi (University Degli Studi di Milano, Milano, Italy). The low-stringency screens were performed by hybridizing the membranes overnight at 42° in 30% formamide, 5 × SSPE (1× = 0.18 M NaCl, 0.01 M sodium phosphate, pH 7.4, 1 mM EDTA), 1% SDS, 5 × Denhardt's solution (1 × Denhardt's = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 150 mg/ml sonicated salmon sperm DNA. The membranes were washed successively in 5 × SSPE and 0.1% SDS, at room temperature, and in 2 × SSPE and 0.1% SDS at 42° for 30 min each. Autoradiography was performed by exposing the membranes to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) for 1-2 days. Clones thus isolated were purified and subjected to dideoxy sequencing using the Sequenase 2 Kit (United States Biochemical, Cleveland, OH). The identity of each clone was then determined by searching for their sequences against the sequences contained in the National Center for Biotechnology Information database using the Blast suite of programs (12). Alignment of the peptide sequences was performed using MacVector (Eastman Kodak) and The Wisconsin Package (Genetics Computer Group, Madison, WI).

Other cDNAs. Chicken 2 cDNA was described previously (13). Rat 6 was kindly provided by Drs. Stephen Heinemann and Jim Boulter. Human 3 was cloned from a human brain library. Chicken 3, 2, and 4 cDNAs were obtained through the generosity of Dr. Marc Ballivet (Department of Biochemistry, University of Geneva, Geneva, Switzerland).

Expression of AChR subunits in X. laevis oocytes. Chicken and human cDNAs were cloned into a modified SP64T expression vector (14) using standard DNA cloning procedures. cRNA was synthesized in vitro using the Megascript kit (Ambion, Austin, TX). Oocytes were defolliculated and injected with either 15 or 100 ng of cRNA per oocyte. Chicken 3, 2, and 4 subunits were expressed by nuclear injections of 2 ng of genomic DNAs per oocyte. The oocytes were incubated in semisterile conditions at 18° in saline solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.6) containing 50% Leibovitz-15 media (GIBCO/BRL, Gaithersburg, MD) buffered to pH 7.4 with 10 mM HEPES. Oocytes were incubated at 18° for 3-6 days before use.

Electrophysiological procedures and drug application. Currents in oocytes were measured using a standard two-microelectrode voltage clamp amplifier (oocyte clamp OC-725; Warner Instrument, Hamden, CT). Electrodes were filled with 3 M KCl and had resistances of 0.5-1.0 M for the voltage electrode and 0.4-0.6 M1/2 for the current electrode. All records were digitized (MacLab/2e interface and Scope software; AD Instruments, Castle Hill, Australia), stored on a Macintosh IIcx computer (Apple Computer, Cupertino, CA) and analyzed using AXOGRAPH software (Axon Instruments, Burlingame, CA).

Drugs used. Epibatidine (oxalate salt) was synthesized at Merck Sharp & Dohme Research Laboratories (Essex, UK) and was a gift from Stephen Fletcher (16). ()-Nicotine tartrate, cytisine, DMPP, and ACh chloride were obtained from Sigma (St. Louis, IL).

	Results

Summary Introduction Materials & Methods Results Discussion References

Isolation and primary structure of the chick 6 AChR subunit. A chicken cochlea cDNA library was screened at low-stringency hybridization conditions using a cocktail of AChR subunit cDNA probes. In addition to identifying cDNAs for 4, 7, 2, and 4 subunits, one 2233-bp cDNA was identified that closely resembled the previously cloned rat 6 AChR subunit (Genbank accession number L08277) as shown in Fig. 1A. The cDNA encodes a predicted mature protein of 464 amino acid residues, preceded by a leader peptide of 30 residues. The sequence contains a cysteine pair homologous to 1 128 and 142 and a second cysteine pair homologous to 1 192 and 193, which identify it as an  subunit. The sequence also contains the four putative transmembrane sequences typical of all AChR subunits. The predicted amino-acid sequence of mature chicken 6 is 86%, identical to that of rat 6 and 88% identical to human 6 (Fig. 1). Most sequence differences occur in the putative large cytoplasmic loop between transmembrane domains three and four, which is the region that typically shows the most variation between species of AChR subunits. Chicken and rat 6 subunits are identical both in parts of the sequence believed to contribute to the ACh binding site (e.g., amino acids of the mature protein 180-200) and in the lining of the cation channel (i.e., amino acids 200-250); therefore, 6 subunits from the two species would be expected to have both similar ligand binding and cation channel characteristics.

View larger version (76K): [in this window] [in a new window]   Fig. 1.   A, amino acid sequence alignment of chicken, rat, and human AChR 6 subunits. B, amino acid sequence alignment of the human, rat, and chicken 4 AChR subunits. Shading, identical residues. Gaps were introduced in the sequences to maximize homologies. Arrows, positions of the predicted leader peptide, cysteine residues conserved in all AChR subunits; arrowheads, cysteine residues conserved in all  subunits; TMD1-TMD4, putative transmembrane domains.

Isolation and primary structure of the human 4 AChR subunit. The deduced amino acid sequence of the human 4 AChR subunit obtained by low-stringency screening of a SH-SY5Y cDNA library is compared with the rat and chicken 4 subunits in Fig. 1B. Protein encoded by the human 4 cDNA has substantial identity with sequences of the chick (75%) and rat (85%) 4 AChRs. A leader peptide, four hydrophobic putative transmembrane domains, and two highly conserved cysteine residues at positions 153 and 167 are characteristic of all -type subunits. Designation of this clone as a  subunit was confirmed by functional tests in which it was shown to form functional AChRs when expressed in combination with human 3 subunits (Fig. 2). An incomplete, nonfunctional human 4 AChR cDNA was published earlier (17).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Chicken 6 AChR subunit mediates detectable function only in combination with the human 4 AChR subunit. Top, currents induced by 300 µM ACh applied as indicated by the bars to oocytes expressing chicken AChRs: left, 34; right, 64. Middle and bottom, responses were recorded from oocytes expressing human 3 and 2 (middle left) or 3 and 4 (bottom left) AChR subunits. Chicken 6 subunits expressed alone or with human 3, 2 (middle right) AChR subunits did not result in detectable responses. However, chicken 6 coexpressed with human 4 (bottom right) consistently produced responses. Currents were obtained 3-5 days after nuclear injection of the cDNAs (chicken 3 and 4) and cRNAs (chicken 6 and human 3, 2, 4). Holding potentials are indicated above the traces in each panel.

Functional expression of the chicken 6 AChR subunit. Multiple attempts to detect functional nicotinic AChRs in X. laevis oocytes injected with in vitro synthesized chicken 6 transcripts either alone or after prior nuclear injection of chick 2, 3, or 4 cRNAs were unsuccessful (Fig. 2). Parallel control experiments with pair-wise coexpression of 2 or 4 together with 3 confirmed the functionality of these cDNAs. Additionally, no functional AChRs were detected when oocytes were injected with mixtures containing 15-100 ng per oocyte of both in vitro synthesized 6 and 4 transcripts (these were obtained by linearizing the chicken 3 cDNA recloned into the NotI site of the pBS SK() vector). Functionality of 4 cRNA was confirmed by successful coexpression with the chicken 4 AChR subunit. In general, maximal currents resulting from expression of the chicken 32 (Fig. 3), 34, and 44 subunit combinations in oocytes clamped at 70 mV did not exceed 500 nA.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Chicken 6 human 4 AChRs, activation by acetylcholine and channel properties. Top left, typical currents induced by application of increasing concentrations of ACh on oocytes coinjected with chick 6 and human 4 cRNAs. Top right, a plot of the dose-response curve for ACh obtained from five oocytes held at 100 mV. Currents were normalized to the maximal current (at 300 µM ACh). Further increase of the ACh concentration induced currents with lower amplitude, suggesting the occurrence of channel blockage by the ACh. Recordings were performed 5 days after injection on an oocyte voltage clamped at 100 mV. Data (mean ± standard error) were fitted with the Hill equation. Middle, potential dependence of chick 6 human 4 AChRs; left, the family of currents induced by application of 300 µM ACh to oocytes expressing 64 held at different potentials from 90 to +30 mV is shown at 20-mV increments; right, a plot of the peak current versus holding potential. The current reverses at 18 mV. Bottom, dependence of the 64 AChR-mediated current on extracellular Ca2+ concentration; left, currents induced at normal Ca2+ concentration (1.8 mM) and after removal of the Ca2+ ions from the perfusion solution (5 mM EGTA was added to chelate possible Ca2+ contamination); right, voltage dependence of the 64 AChR-mediated current in control and in the "0" Ca2+ solution. Voltage ramps were obtained by continuously changing the holding potential from 100 to +50 mV more than 2 sec. Traces are the result of the subtraction of the currents obtained in the absence of the drug from currents during application of 30 µM ACh.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Pharmacological properties of chick 6 human 4 AChRs. Top, inhibition of 64-mediated currents by D-tubocurarine (left) and mecamylamine (right). Curare was coapplied for 6 sec after 4 sec of perfusion of 30 µM ACh. Currents induced by 30 µM ACh before, after 2-min perfusion with 10 µM mecamylamine, and after 10-min washout. Middle left, currents induced by 300 µM ACh before and after 4-sec application of 100 µM nicotine (middle trace) to illustrate the long-lasting antagonism produced by exposure to nicotine. Middle right, currents induced by application of increasing concentrations of (+)-epibatidine are compared with the control current induced by 300 µM ACh. Bottom, a family of concentration-response curves obtained for ACh, nicotine, cytisine, DMPP, and (+)-epibatidine. Averaged data from three to five experiments are presented.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of effectiveness of nicotinic agonists on chick 6 human 4, chick 34, and human 34 AChRs

Functional expression of the rat 6 AChR subunit. Coexpression of the rat 6 subunit, along with the human 4 subunit, resulted in appearance of ACh-induced inward currents that resembled those observed for chick 6 human 4 AChRs (Fig. 5), as expected from the sequence identities of 6 subunits from the two species in both the regions believed to govern ACh binding and channel function (Fig. 1). Currents reversed around 15 mV, and the current/voltage showed strong inward rectification. Rat 6 human 4 AChR-mediated currents were inhibited by curare (n = 4) (Fig. 5). ACh was slightly less potent on rat 6 AChRs (EC₅₀ = 37 µM) compared with chicken 6 AChRs. Nicotine, cytisine, and DMPP behaved as partial agonists with 52%, 30%, and 19% efficacy, respectively, compared with ACh (Fig. 5).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Rat 6 subunits form functional AChRs with human 4 subunits. Top left, a family of currents induced by application of increasing concentrations of ACh to oocytes 5 days after cytoplasmic injection of rat 6 and human 4 AChRs cRNAs. Oocytes were voltage clamped at 100 mV. Top right, a plot of the dose-response curve for ACh obtained from 5 oocytes. Bottom left, inhibition of the rat 6 human 4 AChR-mediated current by the nicotinic antagonist curare. ACh-induced currents before, during, and after, coapplication with 10 µM curare. Bottom right, the maximal currents induced by ACh, nicotine, cytisine, and DMPP are compared. Data are from four oocytes. In each experiment, currents induced by 300 µM agonist were normalized to the current induced by 300 µM ACh (a saturating concentration).

	Discussion

Summary Introduction Materials & Methods Results Discussion References

6 subunits are most closely related in sequence to 3 subunits (~75% amino acid sequence identity) (4, 5). In contrast to 3 subunits, coexpression of the chick 6 subunit with either chick or human 2 subunits did not yield detectable AChR function. It is not clear whether this is an intrinsic attribute of the 6 subunit that discriminates between assembly properties of the 3 and 6 subunit or if it is due to technical shortcomings. Maximal currents detected for the 34 combination were almost two orders of magnitude higher compared with the 64 combination. This might indicate that 6 and 3 subunits differ in assembly affinity for the 4 subunit and/or, possibly, additional subunits are required for more effective functional expression of the 6 subunit. On the other hand, the relatively small amplitudes of the 64-mediated currents and failure to detect function on coexpression with the 2 subunit could reflect levels of 6 expression insufficient for functional detection. We have no independent measure of the quality of the 6 cRNA or the amount of 6 protein produced. The low overall levels of 64 AChR function detected also may reflect inefficient processing or assembly of 6 in X. laevis oocytes compared with the neurons in which 6 might normally be found. There is, as yet, no characterization of 6 protein or function in neurons with which to compare the properties of these cDNAs expressed in X. laevis oocytes.

Despite the very high level of homology between 6 and 3 AChR subunits, they exhibit significant differences in their functional properties. As discussed above, in addition to differences in expression levels and coexpression with 2 and 4 subunits, 64 AChRs exhibit significantly different pharmacological properties compared with either chicken or human recombinant 34 AChRs (Table 1) (16, 18). Therefore, both chicken 6 human 4 AChRs and rat 6 human 4 AChRs retain a unique agonist profile with nicotine, cytisine, and DMPP as poor partial agonists. In contrast, nicotine was shown to be a full agonist for chicken, rat, and human 34 AChRs (Table 1) (16, 18, 19). Nicotine behaves virtually as an antagonist on 64 AChRs. The time course of the nicotine-induced current does not indicate either accelerated desensitization or channel block of the AChRs. Inhibition of 64 AChRs by nicotine strongly resembles the action of nicotine previously described for chicken 32 AChRs but not for 34 AChRs (18). After extensive studies of this phenomenon, these authors concluded that nicotine behaves as a competitive antagonist at low concentrations, but as a partial agonist at higher concentrations, and that its inhibitory action is at least in part contributed by the 2 subunit. Nicotine also behaves as an antagonist for rat homomeric 9 AChRs (20). Epibatidine exhibits extremely high potency for 64 AChRs. High potency of this alkaloid also was described recently for all recombinant and native, chicken, and human 3-containing AChRs (16). Therefore, sensitivity to ACh and epibatidine is similar for 34 AChRs and 64 AChRs, whereas actions of cytisine, DMPP, and especially nicotine clearly distinguish between these two subtypes of AChR. These features could be extremely useful for potential future functional identification of native 6-containing AChRs.

Depletion of Ca²⁺ ions from the extracellular solution resulted in a dramatic decrease of 64 AChR-mediated currents. Similar phenomena were characterized originally for oocyte-expressed and native rat 3 AChRs (21, 22). It was concluded that physiological concentrations of extracellular Ca²⁺ ions enhance neuronal AChR-mediated currents by direct binding on the extracellular side of these AChRs. Alternatively, decrease of the 64 AChR-mediated currents in low Ca²⁺ could be the result of the prevention of activation of a secondary endogenous Ca²⁺-dependent Cl current. This current is known to accompany currents mediated by recombinant AChRs or N-methyl-D-aspartate receptors with relatively high Ca²⁺ permeability expressed in X. laevis oocytes (11, 15, 22). However, the time course of the currents mediated through the oocyte-expressed 64 AChRs suggests that the contribution of the Ca²⁺-dependent Cl current is minimal or nonexistent. A Ca²⁺-dependent Cl current usually is observed as a peak current with a relatively fast inactivation at the beginning of the agonist application and sometimes is misinterpreted as a fast component of desensitization (15, 22, 23).

Preliminary in situ hybridization studies of mRNA expression for the 6 AChR subunit revealed the pattern of distribution of this subunit in developing rat brain (8). Message for the 6 subunit was localized within the medial habenula, locus ceruleus, ventral tegmental area, and substantia nigra compacta. This restricted pattern distribution of 6 message in brain contrasts with the more diverse and diffuse distribution of the 4 (see Refs. 1 and 2 for review) and 7 (24, 25) subunits and rather parallels the distribution of the 3 AChR subunit (1, 26). Now that we have demonstrated that 6 can participate in functional AChRs in combination with 4, the significance of 6 localization in brain will have more effect on our understanding the central effects of ACh, nicotine, and nicotinic drugs. Message for the 4 subunit is colocalized in, but not limited to, the brain areas that contain 6 mRNA (27). Message for 6 RNA is localized in parts of the brain traditionally believed to participate in the rewarding properties of drugs of abuse, with one of the putative mechanisms of addiction involving dopamine release in the neurons of these areas (28). Functional, unidentified, neuronal AChRs were shown to be present in these areas (29-31). The substantia nigra, which degenerates in Parkinson's disease, expresses 6 (8). Nicotinic AChRs are lost in Parkinson's disease (32, 33), and smoking seems to be protective in this disease (34); therefore, AChR subtypes with a limited distribution, including this nucleus, might be useful drug targets for subtype-specific AChR agonists intended for therapy of Parkinson's disease.

With our initial demonstration that 6 can function as part of AChRs formed from subunit cDNAs expressed in X. laevis oocytes, 6 leaves the ranks of orphan subunits and joins the company of numerous potential AChR subtypes that are much better characterized as expressed cDNAs in oocytes than they are in any native neurons. Now the challenge is to detect 6 AChR proteins in neurons, determine their subunit composition, and relate their functional properties to those we have observed in oocytes.