The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function

1.1 Structure of the CHRNA7 gene

The CHRNA7 gene on chromosome 15q13.3 has 10 exons, differing from other nicotinic receptor subunits, which only have six. We cloned the human gene from postmortem brain (Genbank U40583) and discovered that the gene is partially duplicated [45]. Exons 5-10 are duplicated, along with a large cassette of DNA (∼300kb). The duplication interrupted a second partial duplication of another gene, and maps centromeric to CHRNA7 by 1.6Mb [45-47]. The chimeric gene CHRFAM7A is complex (Figure 1). Upstream of Exons 5-10 of CHRNA7 (red), are three exons from a partial duplication of ULK4 (C,B,A;blue), a serine/threonine kinase gene mapping to 3p22.1. 5′ of these three exons is an additional exon of unknown provenance (D; pink). CHRNA7 DNA sequence in CHRFAM7A is 99.9% conserved [45]. CHRFAM7A is expressed in human brain at low levels (approximately one order of magnitude less than CHRNA7 in hippocampus), but is expressed abundantly in peripheral lymphocytic cells and in multiple other tissues. While a full phylogenetic study has not yet been done, CHRFAM7A is not found in closely related primates [48] and does not appear to be found in rodents (unpublished data). Thus, the duplication is recent. This may not be surprising as CHRNA7 is one of the oldest ligand-gated ion channel genes and phylogenetic duplication may have contributed to the origin of this large gene family [49-51]. CHRFAM7A was recently shown to have a regulatory role that, thus, is unique to humans [52].

1.2 Copy number variation in the chromosome 15q13.3 region

Reports of large copy number variations (CNV) on 15q13.3 that include the CHRNA7 gene suggest that CNV in this gene cluster is associated with schizophrenia [44;53]. Examination of these reports of duplications and deletions at 15q13.3 show that in many cases CHRNA7 has been deleted, but the partial duplication, CHRFAM7A is present. It has not been known whether copy number of CHRFAM7A could be important. There are also smaller insertions and deletions within the CHRNA7/CHRFAM7A gene cluster. Approximately 10% of individuals have only one copy of CHRFAM7A. This CNV has an allele frequency of 0.08 in Caucasians and 0.12 in African Americans (not significantly different). Rare individuals are missing both copies [45;54]. Again, a common CNV for CHRFAM7A would not be unusual if it is a new duplication.

1.3 Mutations in the CHRNA7 and CHRFAM7A genes in schizophrenia

Mutation screening in exons and splice junctions of the CHRNA7 gene and its partial duplication, CHRFAM7A revealed no mutations in the coding region of the full-length gene, CHRNA7, that were associated with schizophrenia [45;55]. Therefore, the receptors from CHRNA7 that are expressed in the disorder have a normal amino acid sequence. However, we did find mutations in the proximal promoter region of the CHRNA7 gene that functionally reduce transcription and are associated with both schizophrenia and the P50 deficit [42]. The promoter mutations are more prevalent in schizophrenic non-smokers and may account for the low levels of CHRNA7 mRNA in that group [56]. One of these mutations, rs3087454, has recently been associated with a pharmacogenomic response to DMXB-A, an α7*nAChR partial agonist [57].

We also found a 2bp deletion in exon 6 that is found only in the CHRFAM7A gene [55]. The overall allele frequency of this mutation, CHRFAM7AΔ2bp is 0.22. To evaluate the 2bp deletion in exon 6 of CHRFAM7A, the CNV must be measured in addition to screening for the polymorphism, since ∼10% of individuals have only one copy. We have recently completed such a study and find that the 2bp deletion in exon 6 of CHRFAM7A is associated with schizophrenia [54]. Others have found association of the 2bp deletion to schizophrenia [58] or to sensory processing deficits such as the P50 deficit and antisaccade performance [59-61].

The 2bp deletion in CHRFAM7A is also associated with a gene inversion [47]. The wild type allele is in a head-to-head orientation with respect to the full-length CHRNA7 gene, but CHRFAM7AΔ2bp is oriented in the same direction. CHRFAM7AΔ2bp is more common in Caucasians than in African Americans, suggesting that the inversion of the gene occurred simultaneously or shortly prior to the 2bp deletion [54]. The functions of these two versions of the gene are not known.

There is a common, but synonymous, mutation in CHRFAM7A at position 654bp. Of the Caucasian individuals that have been genotyped for both the 2bp deletion and the SNP at 654bp, the two mutations appear to be in linkage disequilibrium. However, more subjects need to be genotyped to confirm this finding. An additional common mutation is non-synonymous, 1466bp C→T, resulting in an amino acid change of a serine to a leucine at aa 489. This amino acid would be in the cytosolic tail. Although a function for this S489L mutation has not yet been defined, a similar mutation that changed a glycine to a serine in CHRNA7 resulted in phosphorylation differences and alterations in receptor function [62].

1.4 The CHRNA7 gene is regulated differently in schizophrenic smokers, compared to control smokers

A conundrum concerning the expression of the CHRNA7 gene product, α7*nAChR, exists in schizophrenia. In a microarray comparison of gene expression in postmortem hippocampus of control and schizophrenic smokers and non-smokers, we found that the CHRNA7 gene is differentially regulated at the mRNA and protein level in schizophrenic smokers [35;56]. Schizophrenic non-smokers have low levels of CHRNA7 mRNA and protein, compared to controls, but schizophrenic smokers have mRNA and protein levels for CHRNA7 similar to control smokers. Thus, in schizophrenic smokers there is adequate mRNA and protein [56]. These results are not consistent with the low levels of [¹²⁵I]-α-bungarotoxin and [³H]-methyllycaconitine binding seen in human postmortem brain of schizophrenic subjects [2;39;40]. The data are consistent, however, with an assembly, trafficking, or binding site defect. In the current report we investigated the possibility that the gene duplication, CHRFAM7A regulates the expression and function of CHRNA7.

2.1 Cloning of CHRFAM7A and CHRFAM7AΔ2bp

Generation of a full-length cDNA clone of the duplicated gene, CHRFAM7A, was performed by PCR cloning. The exon D –3′ UTR cDNA was generated by PCR amplification of reverse transcribed mRNA derived from human primary lymphocytes, which abundantly express CHRFAM7A. Primer sequences were 5′-CTCGGTGCCCCTTGCCATTT-3′ (Sense) and 5′-CCTTGCCCATCTGTGAGTTTTCCAC-3′ (antisense) and were located at position 140 and 1850 from the start of exon D. PCR was performed using taq/pfu polymerase mix with supplied buffers and reagents (Stratagene, La Jolla, CA). After TA cloning, the fragment was moved to pcDNA3.1 (Invitrogen, Carlsbad, CA), a mammalian expression vector.

The 2bp deletion in exon 6 of CHRFAM7A was introduced into pcDNA3.1 CHRFAM7A by PCR with the QuikChange II XL Site-Directed Mutagenesis Kit, according to the manufacturer's protocols (Stratagene, La Jolly, CA). All clones were sequenced bidirectionally using internal primers and dRhodamine dye terminators on an ABI 3100 Avant DNA sequencer (Applied Biosytems, Inc., Foster City, CA) for confirmation of correct sequence. Clones were designated as pcDNA3.1-CHRFAM7A and pcDNA3.1-CHRFAM7AΔ2bp.

An α7 full-length clone was also constructed as a control. Primer sequences were 5′-CGCTGCAGCTCCGGGACTCAACATG-3′ (sense) and 5-TGCCCATCTGTGAGTTTTCCA CATG-3′ (anti-sense) and were located at position (from start ATG) (-22) and +1638 respectively. PCR fragments were gel purified and TA cloned into the pcDNA3.1 mammalian expression vector (Invitrogen)(pcDNA3.1-CHRNA7).

From the original exon D-10/pcDNA3.1 and α7/pcDNA3.1 cDNA constructs, PCR primers were designed to amplify α7-in frame sequences for expression with a V5 epitope; the 5′ end of the antisense primer terminated just prior to the normal stop codon in exon 10. PCR fragments were purified from 1% agarose and TA cloned into the pcDNA3 Topo/V5 vector (Invitrogen, Carlsbad, CA), transformed, isolated, prepped and sequenced as described above. The clone is designated as pcDNA3.1-CHRFAM7A-V5.

2.2 Cell Culture and Transfections

SH-EP1 cells were used for heterologous expression studies. SH-EP1 are a non-neuronal epithelial cell line derived from the neuroblastoma cell line SH-SY5Y [63]. Wild-type SH-EP1 cells do not express endogenously detectable α7 mRNA or protein. Cells were cultured in DMEM media supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 1 mM sodium pyruvate, 4 mM L-glutamine, and 100 U/ml penicillin/streptomycin (Gibco Life Technologies/ Invitrogen, Rockville, MD) in 5% CO₂ at 37°C.

Optimal transfection conditions were determined empirically using Green Fluorescent Protein (Invitrogen). Cells (150,000) were seeded in 12-well plates containing 12 mm poly-L-lysine-coated glass coverslips (Becton-Dickinson, Franklin Lakes, NJ). Transfections were performed 24 hours after plating using Superfect reagent (Qiagen, Valencia, CA). Briefly, 5-10 μg of DNA was added to an unsupplemented D-MEM in final volume of 150 μl. Superfect (60 μl) was then added and the mixture was allowed to incubate for 10 minutes at room temperature before the addition of 1 ml of complete media. During DNA complex formation, cells were washed once with phosphate-buffered saline (PBS) and the DNA/Superfect mix was added to the cells. Cells were then incubated for 2 hours before the transfection reagent was removed. Cells were washed twice with PBS and returned to the incubator with complete media for 48 hours. Forty-eight hours post-transfection, cells were fixed in 4% paraformaldehyde, washed with PBS and permeabilized in successive washes of 10, 95 and 100% ethanol. After washing, coverslips were blocked for 20 minutes in complete media. Cells were then blocked using 4% goat serum (containing 2% bovine gamma globulin and 0.3% Triton-X100) at room temperature for 1 hour. Primary antibodies were mouse anti-V5 (Invitrogen) diluted 1:100 and rabbit anti-α7 (kind gift of Cecilia Gotti, University of Milan) diluted 1:1000. Cells were incubated for 1 hour, washed 3× with PBS and secondary antibodies applied. Secondary antibodies were donkey-anti-mouse Cy2 (1:1000) and donkey anti-rabbit tetramethyl rhodamine (TRITC) (1:1000) (Jackson Immunochemicals, West Grove, PA) and were incubated for 1 hour at room temperature. All coverslips were then washed 3× with PBS, mounted and visualized using appropriate filters on a Microphot-FXA fluorescence microscope (Nikon, Melville, NY).

2.3 Oocyte expression

Oocytes were isolated and selected as previously described [64]. Briefly, oocytes were harvested from mature Xenopus laevis females, anesthetized with tricaine. Frogs were sacrificed following ethical animal protocols in Geneva, Switzerland. Oocytes were isolated by mechanical and enzymatic action using type I collagenase (Sigma, Basel Switzerland) at 0.2% in a medium deprived of calcium. Stage V and VI oocytes were placed in 96 wells microtiter plates (NUNC) in BARTH medium that contained 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 10 mM HEPES, 0.82 mM MgSO₄·7H₂O, 0.33 mM Ca(NO₃)₂·4H₂O, 0.41 mM CaCl₂·6H₂O, adjusted to pH 7.4 with NaOH and supplemented with kanamycin (20 mg/ml), penicillin (100 U/ml) and streptomycin (100 U/ml) (Sigma). Nuclear injections of 2 ng of cDNA expression vectors, containing equal molar ratios of the genes of interest were performed with an automatic injection system [65]. Cells were subsequently maintained in Barth's medium at 18 °C.

2.4 Recording of α7* receptor properties

Electrophysiological properties of oocytes were determined 2 to 5 days after injection with an automated system equipped with a two electrode voltage clamp (Geneclamp Axon Instrument, Molecular Devices, Sunnyvale, CA). Oocytes were continuously superfused with OR2 (control medium) that contained 82.5 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, adjusted to pH 7.4 with NaOH. Current was generated by 5s pulses of 200 μM acetylcholine (ACh). Data were filtered at 10 Hz and digitized at 100 Hz with an analog to digital converter (National Instruments, Austin, TX) and stored on a personal computer. Data acquisition, treatment and statistics were performed using Matlab (MathWorks Inc., Natick, MA).

2.5 [I¹²⁵]-α-Bungarotoxin assay

Injected oocytes generating a current were selected and were pre-incubated in a solution of OR2 with 2%BSA for 10-20 min. Oocytes were then incubated in 50 μl of OR2-2%BSA with 10nM [I¹²⁵]-α-bungarotoxin (Institute of Isotopes Co., Budapest, Hungary) for 1h at 18°c. After 3 washes with OR2-2%BSA, the radioactivity of each oocyte was measured with a scintillation counter and normalized to non-injected oocytes.

2.6 RNA isolation from injected oocytes and QRT-PCR quantification of CHRFAM7A and CHRNA7

Oocytes were placed in 1 ml RNAlater (Ambion, Austin, TX) at 4°C for >24 hour. Ten eggs were homogenized for 30 seconds in 0.5ml of Trizol (Invitrogen, Carlsbad, CA) using a Pestle Grinder (Fisher Scientific, Pittsburgh, PA). The homogenate was placed on ice for one minute, then room temperature for 5 minutes before extraction with chloroform. Following centrifugation at 12,000rpm for 15 minutes at 4°C, the aqueous layer was removed to a new tube. Purification of RNA was done using an RNeasy mini column (Qiagen, Valencia, CA) using manufacturer's protocol. Yield was 0.5-1 ug total RNA per oocyte.

cDNA was synthesized using Superscript 3 (Invitrogen) according to manufacturer's directions. Real-time PCR was performed using human CHRNA7 primer sets (Forward 5′-TTTACAGTGGAATGTGTCAGA-3′, Reverse 5′-TGTGGAATGTGGCGTCAAG-3′), CHRFAM7A (Forward 5′-TGGATAGCTGCAAACTGCGA-3′, Reverse 5′-TACTGGCAATGCCCAGAAGA-3′) and GAPDH (5′-Forward GGTATCGTGGAAGGACTC-3′, Reverse 5′-GGATGATGTTCTGGAGAGC-3′), using iQ SYBR Green (Biorad, Hercules, CA). All three real-time assays were run for 40 cycles; 94°C for 15 sec, 58°C for 30 sec, 72°C for 30 sec.

3.1 Structure of the CHRNA7/CHRFAM7A gene cluster

The partial duplication of the CHRNA7 gene is recent, not being found in primates or rodents [48]. The relationship of the two genes is shown in Figure 1A. They are approximately 1.6 Mb apart. The region between the CHRFAM7A and CHRNA7 contains two other genes TRPM1 and KLF13. A detailed study, utilizing tagged SNPs across the linkage region at 15q13.3 shows that there is broad association with schizophrenia at this locus [66].

The chimeric gene, CHRFAM7A, contains exons 5-10 of the full-length CHRNA7 gene, exons A, B, and C duplicated from ULK4, and exon D of unknown provenance (Figure 1A). Exon sizes were determined: A, 47bp; B, 63bp; C, 125bp; D, 297bp [45] and the gene was registered in GenBank (AF029837). Mutation screening of the coding regions and exon/intron borders in both CHRNA7 and CHRFAM7A was previously reported [55]. Non-synonymous mutations were rare in CHRNA7 and were not associated with schizophrenia. Therefore, the receptors from CHRNA7 that are expressed in the disorder have a normal amino acid sequence.

Several mutations were mapped specifically to CHRFAM7A (Gault et al., 2003). A 2bp deletion in exon six of CHRFAM7A (CHRFAM7AΔ2bp) was found. The mutation is common; the overall allele frequency of this mutation is 0.22, and it is more prevalent in Caucasian individuals than in African Americans [55]. The CHRFAM7A gene also varies in copy number. Approximately 30% of individuals have only one copy and 5% have no copies. Thus to evaluate the 2bp deletion in exon 6 of CHRFAM7A, copy number variation must be measured in addition to screening for the polymorphism. We have recently completed such a study and find that the 2bp deletion in exon 6 of CHRFAM7A is associated with schizophrenia in both Caucasians and African Americans [54]. Others have found association of the 2bp deletion to schizophrenia [58] or to sensory processing deficits such as the P50 deficit and antisaccade performance [59;60]. The 2bp deletion may change the structure of the protein translated from the mRNA.

3.2 CHRFAM7A is expressed in transfected cells

A cDNA clone of CHRFAM7A with a V5 peptide tag (pcDNA3.1 CHRFAM7A-V5) was transfected into SH-EP cells, plated on cover slips. An anti-V5 antibody (Invitrogen) was utilized to detect protein product. Figure 2A shows a positive V5 signal, indicating that the peptide product was transcribed and translated, although it is not abundantly expressed in the transfected cells. An α7 polyclonal antibody to the large cytoplasmic loop of the protein was used in 2B, labeled with rhodamine. A positive signal and overlap in 2C shows that the translated peptide contains sequence for the α7 subunit.

3.3 Translation of CHRFAM7A mRNA

Several initiator methionines in exon D could result in truncated transcripts that do not contain CHRNA7 coding sequence. Use of an initiator methionine in exon B of CHRFAM7A would produce a protein including amino acids coded by part of exon B, exon A, and exons 5-10 of CHRNA7. The peptide would include the disulfide bridge and vicinal cysteines contained in the amino terminus of the α7 subunit. A putative glycosylation site would be lost and part of the agonist binding site (Figure 1C). If the 2bp deletion in exon 6 is present, use of the same initiating methionine would code for a peptide with exon 6 amino acid sequence to the deletion. At that point a frame shift would result in 40 unique amino acids before a stop codon was reached. There are, however, two ATG codons in exon 6. A translation start at one of these would terminate within a few amino acids unless the 2bp deletion was present. Deletion of these two base pairs would allow translation of a peptide with considerable α7 sequence. The peptide would be out of frame until the 2bp were reached, coding for either 6 or 13 amino acids, depending on which ATG was used. At that point, the frame shift caused by the 2bp deletion would return the amino acid sequence to that of the α7 subunit. The peptide resulting from translation of mRNA without the 2bp, initiating in exon 6, would be missing the signal peptide and nearly the entire binding site, but would contain all of the CHRNA7 membrane-spanning regions.

3.4 The CHRFAM7A gene product is a dominant negative regulator of α7*nAChR function

The CHRFAM7A gene is expressed in both human brain and in the periphery. To study function of this chimeric gene product and effects of the 2bp deletion in exon 6, we expressed clones of the genes alone and together with a full length CHRNA7 cDNA in oocytes. Xenopus laevis oocytes were isolated and injected as described. A total of 2ng of cDNA, containing molar equivalent amounts for each gene were injected. Three injection groups were utilized: pcDNA3.1-CHRNA7 + vector, pcDNA3.1-CHRNA7 + pcDNA3.1-CHRFAM7A, and pcDNA3.1-CHRNA7 + pcDNA3.1-CHRFAM7AΔ2bp. Two to five days after injection, oocytes were superfused with control medium and current, generated by 5s pulses of 200 μM acetylcholine (ACh), was measured.

Determination of the ACh-evoked current in a large population of oocytes obtained from different batches revealed that expression of the duplicate gene caused a reduction of the response amplitude (Figure 3A). Currents were reduced by 53% when pcDNA3.1-CHRFAM7A was included. The presence of the 2bp deletion in exon 6, pcDNA3.1-CHRFAM7AΔ2bp, further reduced the current amplitude of the full-length gene product by an additional 10%. The difference in current amplitude is best evidenced by plotting the histogram of current distribution for pcDNA3.1-CHRNA7 + pcDNA3.1, pcDNA3.1-CHRNA7 + pcDNA3.1-CHRFAM7A, and pcDNA3.1-CHRNA7 + pcDNA3.1-CHRFAM7AΔ2bp as shown in Figures B, C and D, respectively.

3.5 Coexpression of CHRNA7 and CHRFAM7A does not alter transcription from either gene

Total RNA was isolated from oocytes injected with cDNA coding for CHRNA7 and vector or cDNA for CHRNA7 and either CHRFAM7A or CHRFAM7AΔ2bp. Real-time QRT-PCR was utilized to quantify message for CHRNA7 and CHRFAM7A in RNA isolated from each preparation, with primers specific for each gene. Mean normalized expression was calculated from the control values for the gene of interest and the housekeeping gene, GAPDH, and the amplification efficiencies [67]. As shown in Table 1, expression of the gene duplication, CHRFAM7A did not alter the transcription of CHRNA7.

3.6 Presence of the duplicated gene product from CHRFAM7A, changes the [¹²⁵I]-α-bungarotoxin binding properties of α7*nAChR

Oocytes were injected with cDNA for CHRNA7+pcDNA3.1, CHRNA7+CHRFAM7A, or CHRNA7+CHRFAM7AΔ2bp at a final concentration of 2ng/μl. After 5-7 days, the amplitude of the current elicited by brief pulses (5 s) of ACh (200 μM) was measured as described in Methods. Oocytes responding to the ACh test pulse were incubated in OR2-BSA with 10nM [¹²⁵I]-α-bungarotoxin (I-BTX) for 1h. Cells were then thoroughly washed to remove unspecific binding and the radioactivity in each oocyte was measured in a scintillation counter. Non-specific binding was determined by measuring the average I-BTX binding on non-injected oocytes. The histograms of I-BTX binding obtained after subtraction of the non-specific binding obtained for CHRNA7, CHRNA7+CHRFAM7A and CHRNA7+CHRFAM7AΔ2bp are shown in Figure 4A and suggest that cells expressing the duplicate might have a smaller number of binding sites at the cell surface. As there was less current for equivalent binding this is consistent with the hypothesis that the assembly of a truncated subunit, such as the chimeric CHRFAM7A or CHRFAM7AΔ2bp with the CHRNA7 gene product produces a receptor that is less functional. A plot of the peak current versus counts (CPM) yielded linear relationships that were readily fitted by linear regression with high correlation coefficients (CHRNA7+pcDNA3.1, R²=0.995; CHRNA7+CHRFAM7A, R²=0.867; CHRNA7+CHRFAM7AΔ2bp, R²=0.915 (Figure 4B).

3.7 Potentiation of CHRNA7 and CHRFAM7A by the allosteric modulator PNU-120596

PNU-120596 is a potent allosteric modulator of the α7*nAChR causing an increase of the maximal amplitude of the ACh-evoked current, reduction of the EC₅₀ and reduced desensitization [68]. Previous work suggested that co-expression of the duplicate gene could alter the allosteric modulation of the receptor [52]. To understand the possible relevance of such an observation it must be recalled that the absence of potentiation by PNU-120596 observed in the α7-5HT3 chimera suggested that this molecule must interact with the transmembrane domain [69]. As the duplicate is thought to have a different N-terminal domain from the α7 subunit, but has the same amino acid sequence for the transmembrane domains, PNU-120596 potentiation in the presence of the duplicate in the receptor complex might be expected. To assess the hypothesis, PNU-120596 effects were examined at CHRNA7, CHRNA7+CHRFAM7A and CHRNA7+CHRFAM7AΔ2bp, injected oocytes. Cells injected with an equivalent concentration of the cDNAs encoding the two genes showed non-significant differences in the magnitude of potentiation measured as the ratio of the ACh-evoked current observed after PNU-120596 versus the current evoked by the same ACh test pulse (1280 μM, 5 s) in control (Figure 5). As shown in Figure 5A, exposure to 10 μM PNU-120596 for 30 s causes a dramatic increase in the amplitude of the ACh-evoked current that is accompanied by a significant increase in the response duration that was interpreted as a reduction of desensitization [68]. It could be argued that only receptors composed of CHRNA7 are potentiated by PNU-120596 and this effect might mask the fraction of receptors that include the duplicate. To address this hypothesis additional experiments were designed in which the ratio of CHRNA7 versus the duplicate gene was reduced to 1:10. Currents evoked by a brief pulse of 1280 μM ACh for 5 seconds were measured in control and following 30 seconds exposure to 10 μM PNU-12056. Average currents measured on the same day in sibling oocytes were respectively 5.8 ± 0.66 μA for CHRNA7, 0.9 ± 0.33 μA for CHRNA7+CHRFAM7A and 1.1 ± 0.19 μA for cells injected with CHRNA7+CHRFAM7AΔ2bp. These data are in agreement with previous experiments indicating that expression of the duplicate gene causes a reduction of the average ACh-evoked current [52]. However, a different picture emerged when examining the amplitude of the ACh-evoked current after PNU-120596 exposure. Peak inward currents were of 28.0 ± 3.3 μA for CHRNA7, 12.8 ± 1.23 μA for CHRNA7+CHRFAM7A and 22.0 ± 1.77 μA for cells injected with CHRNA7+CHRFAM7AΔ2bp. The potentiation caused by PNU-120596 is best observed when plotting the ratio of these values as shown in Figure 6A.

Determination of the concentration activation curves in control and following PNU-120596 exposure revealed no major difference in sensitivity between the α7 cells and those expressing the duplicate gene. In control conditions the EC₅₀s and Hill Coefficients were: 38 ± 3 μM, nH = 1 for CHRNA7; 105 ± 18 μM, nH = 0.8 for CHRNA7+CHRFAM7A; 47 ± 8 μM, nH = 0.9 for CHRNA7+CHRFAM7AΔ2bp. Following exposure to PNU-120596 the values were: 3.6 ± 0.1 μM, nH = 5 for CHRNA7; 5 ± 0.9 μM, nH = 3 for CHRNA7+CHRFAM7A; 3.6 ± 0.3 μM, nH = 3 for CHRNA7+CHRFAM7AΔ2bp. As expected, exposure to the positive allosteric modulator caused a shift to the left of the ACh sensitivity accompanied by an increase in the Hill coefficient. This suggests that while expression of the duplicate gene causes a reduction of the ACh current amplitude it does not significantly affect the sensitivity of the receptor to its natural ligand.