Recent reports demonstrate that the RIC-3 (resistant to inhibitors of cholinesterase-3) protein is important for the maturation of nAChRs (nicotinic acetylcholine receptors). In the present study RIC-3e, a novel variant of RIC-3, is described. This variant contains a deletion of exons 4 and 5 of RIC-3, resulting in a protein product lacking a conserved coiled-coil domain. Like RIC-3, the new variant is predominantly, but not exclusively, expressed in the brain. The analysis of expression of variant RIC-3 mRNA and of α7-nAChR mRNA in a set of human tissues shows a similar profile. The RIC-3e protein is functionally active and enables surface expression of mature α7-nAChRs in cell lines not otherwise permissive for the expression of this receptor.
nAChRs (nicotinic acetylcholine receptors) are ligand-gated ion channels which are widely distributed in the central and peripheral nervous system. They are involved in a variety of physiological processes including cognition and development, and their dysfunction has been associated with a variety of neurological disorders [1–5]. Hence nicotinic receptors represent important and intensively studied targets for therapeutic intervention. The α7-nAChR is of particular relevance for Alzheimer's disease and schizophrenia, but studies focusing on this receptor are challenging since it cannot be easily expressed in heterologous systems [6–9]. In fact, the complex process leading to the functional expression of α7-nAChR is likely to require the activity of multiple cell-specific assembly and trafficking proteins [10,11]. It was shown previously that the protein RIC-3 (resistant to inhibitors of cholinesterase-3) is necessary for the functional expression of α7-nAChRs [12,13]. Human RIC-3 belongs to a conserved gene family  and is predicted to contain a cleavable signal peptide sequence in vertebrate species, followed by a proline-rich domain, one transmembrane domain and a cytoplasmic component that consists of a conserved coiled-coil domain and a non-conserved C-terminus . Further experimental evidence suggests the absence of a signal peptide, indicating that RIC-3 has two transmembrane domains . The function of this protein is not yet clear, but the prevailing hypothesis states that RIC-3 acts in the ER (endoplasmic reticulum) as a chaperone which regulates the expression of nicotinic and 5-HT3 (5-hydroxytryptamine-3) receptors by either promoting their maturation and functional expression or by retaining the receptors in the ER, depending on the composition and type of receptor [17,18]. The precise mechanism of action of this protein is not known; however, experimental evidence supports an interaction of RIC-3 with unassembled nAChR subunits . To date, the existence of four distinct transcripts of human RIC-3 has been demonstrated , but little is known about the function of these variants.
In the present study we report the identification, cloning and detailed pattern of expression of a new variant of human RIC-3, and demonstrate that this variant is able to promote the maturation and surface expression of α7-nAChRs in cells that are not otherwise permissive for the expression of this receptor.
MATERIALS AND METHODS
PCR amplification was carried out using a proofreading enzyme (Phusion High-Fidelity DNA Polymerase; Finnzymes) following the manufacturer's instructions. DMSO [3% (v/v)] was added to the reactions. The oligonucleotide primer sequences used were as follows: 5′-CACCATGGCGTACTCCACAGT-3′ (forward) and 5′-TCACTCTAAACCCTGGGGGT-3′ (reverse). Reaction details were as follows: 98°C for 1 min; 98°C for 30 s, 65°C at 30 s (decreasing by 1°C every cycle) and 72°C for 30 s for 10 cycles; 98°C for 30 s, 55°C for 30 s and 72°C for 30 s for 30 cycles and 72°C for 7 min. cDNA from adult human hippocampus (see below) was used as the DNA template. The PCR products were purified using a Qiagen Gel Extraction kit following the manufacturer's instructions, cloned into pCR-Blunt II-TOPO (Invitrogen) and subcloned into pcDNA3.1 Zeo(+). The constructs were then DNA sequenced (MWG Biotech). The alignment of the amino-acid sequences was performed using MUSCLE 3.41 [18a] and visualized with GeneDoc 2.6.002 (http://gendiapo.sourceforge.net/).
Real-time qPCR (quantitative PCR)
cDNA was synthesized from 2 μg of polyadenylated RNA (Clontech) employing Superscript II reverse transcriptase (Invitrogen) and an oligo(dT) primer (Proligo France SAS) according to the manufacturer's instructions. Real-time PCR was performed using an I-Cycler and the iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions. cDNA (1 μl) was used as a template and the primer concentration used was 10 μM. Reactions were carried out in triplicate and reaction conditions were as follows: 95°C for 5 min, followed by 95°C for 30 s and 55°C for 30 s for 45 cycles. As a control, amplification without a template was performed. The fluorescence Ct (threshold cycle value) was determined using the iCycler iQ real-time PCR detection system software (Bio-Rad). The relative starting quantities of each transcript were determined by interpolating the Ct values of the unknown samples to the standard curve. Real-time PCR experiments were repeated twice for each target sequence. Quantification of β-actin expression was carried out in parallel in order to normalize the levels of expression in each sample. Oligonucleotide primer sequences were designed against different exons of human RIC-3, RIC-3e and α7-nAChR genes. Primer sequences were as follows: 5′-CAGATCATTTGGGTTGGGAAA-3′ (forward) and 5′-CACACGAGGTAACAGAATTATCTTCCT-3′ (reverse) to amplify a product located within exon 6 of RIC-3; 5′-GGGAAAACAACTGCAGAGGAT-3′ (forward) and 5′-TCTTCAGGGTAACTAATTTTCCTG-3′ (reverse) to amplify specifically the new variant RIC-3e; 5′-GCAAGAGGAGTGAAAGGTTCTATGA-3′ (forward) and 5′-CATGGTCACTGTGAAGGTGACA-3′ (reverse) to amplify a product within human α7-nAChRs; 5′-CCTGGCACCCAGCACAAT-3′ (forward) and 5′-GCCGATCCACACGGAGTACT-3′ (reverse) to amplify a fragment within human β-actin.
Cell culture and transfection
CHO-K1 cells were cultured in Ham's F12 medium with 10% (v/v) FBS (fetal bovine serum), and HEK-293 cells (human embryonic kidney cells) were cultured in DMEM (Dulbecco's modified Eagle's medium) with 10% (v/v) FBS. All media were supplemented with 2 mM Glutamax, 100 units/ml penicillin and 100 μg/ml streptomycin. All media and supplements for cell culture were from Gibco, unless specified otherwise. For transient transfections, 7×104 cells/cm2 were plated one day before transfection on 13-mm-diameter coverslips treated with 10 μg/ml poly-D-lysine. For FLIPR (fluorescence imaging plate reader) Ca2+ measurements, HEK-293 cells were plated in 96-well clear-bottomed black-well plates (Costar) at a density of 4×104 cells/well and cultured for 24 h prior to transfection. All transfections were carried out using Lipofectamine™ 2000 (Invitrogen) following the manufacturer's instructions. DNA (0.1 μg/cm2) was used for each transfection. The ratio of DNA/Lipofectamine™ 2000 used for transfection was 1:3 (w/v). The transfection mixture was prepared in OptiMEM, and the incubation time with the transfection mixture was 4 h. After transfection, fresh medium was added and cells were incubated at 37°C. Assays were performed 24–48 h after transfection. All manipulations with cells were carried out in sterile conditions. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
α-Bgt (α-bungarotoxin) fluorescent staining
For fluorescence microscopy, HEK-293 and CHO-K1 cells were cultured on poly-D-lysine-coated glass coverslips at a density of 7×104 cells/cm2. Labelling was performed using 0.5 μg/ml Alexa Fluor® 555-conjugated α-Bgt (Molecular Probes) for 45 min at 4°C. After three washes with PBS, cells were fixed with 3% (w/v) paraformaldehyde in PBS for 15 min at room temperature (25°C). Coverslips were subsequently mounted with Prolong® Gold Antifade reagent (Molecular Probes) on glass microslips and analysed with a Zeiss LSM510 confocal microscope equipped with Argon and He/Ne lasers.
2+ flux measurements using a FLIPR system
FLIPR-Ca2+ experiments were performed as described previously . Briefly, 24 h post-transfection, the medium was replaced with 100 μl of assay buffer [Hanks balanced salt solution and 20 mM Hepes (pH 7.4)] containing 4 μM Fluo-4-AM (Invitrogen), 0.02% pluronic acid and 5 mM probenecid (Sigma). After incubation for 30 min at 37°C, the labelling solution was removed and 200 μl of assay buffer containing 2.5 mM probenecid and 10 μM MLA (methyllycaconitine) (Tocris Bioscience) was added where appropriate. Plates were then transferred to a FLIPR system (Molecular Devices) with excitation and emission wavelengths set to 488 nm and 540 nm respectively. Basal fluorescence was recorded for 30 s, followed by the addition of 50 μl of a 5× nicotine (Sigma) or PNU-120596 (Tocris Bioscience) solution and measurement of fluorescence for 10 min. Subsequently, 50 μl of a 6× solution of nicotine was added and fluorescence was measured for 3 min. Measurements were made at 1 s intervals for 1 min after the addition of the compound and at 30 s intervals for the remaining time. The results were extrapolated from the FLIPR raw data as the maximum minus the minimum fluorescence signal intensity in two intervals corresponding to the first and the second additions of compounds.
RESULTS AND DISCUSSION
Identification of a novel variant of human RIC-3
In order to clone RIC-3 transcripts, PCR was carried out using cDNA from adult human hippocampus as a template and a proofreading enzyme. The primers used in the reactions were designed based on the known human RIC-3 sequence (GenBank® nucleotide sequence database accession number NM_024557) and were composed of the initiation and stop codons. Four distinct products were obtained from reverse-transcription PCR (Figure 1). The specificity of the primers employed was confirmed by performing the reactions with single primer controls; also, a negative control PCR lacking template cDNA did not result in any amplification (results not shown). The band of approx. 1100 bp (Figure 1) corresponds to the major form of RIC-3 (GenBank® nucleotide sequence database accession number AY326435) , as confirmed by DNA sequencing. Four human RIC-3 variants (a, b, c and d) have been described previously . RIC-3a is the major sequence of RIC-3; for simplicity, we will refer to this splice variant as RIC-3. On the basis of the apparent length of the RIC-3 DNA sequence, the two faster migrating products most probably correspond to the shorter variants of RIC-3 (GenBank® nucleotide sequence database accession numbers AK021670 and AL832601) . The minor product of approx. 900 bp does not correspond to any of the RIC-3 variants identified previously. This putative human RIC-3 variant was cloned into the pCR-Blunt II-TOPO vector and sequenced. This sequence (GenBank® nucleotide sequence database accession number AM422214) demonstrates that the cloned fragment corresponds to human RIC-3. We named this novel variant RIC-3e, following the nomenclature proposed by Halevi et al. . The initiation and stop codons are the same as those reported previously for RIC-3 . A comparison with the gene structure of the human RIC-3 locus shows that RIC-3e contains exons 1, 2, 3 and 6. The deletion of 240 bp corresponds to the lack of exons 4 and 5, probably as a result of alternative splicing. The prediction of the amino-acid sequence and alignment with the known human RIC-3 variants confirmed that the novel cDNA carries a deletion of 240 bp and encodes an RIC-3 protein of 288 amino acids which lacks the coiled-coil domain and part of the C-terminal sequence (Figure 2).
Molecular cloning of a novel variant of
Amino-acid sequence of RIC-3e and comparison with known variants
Expression profile of
RIC- 3 variants and α 7- nAChR in human tissues
Little is known about the tissue-specific distribution of RIC-3. Studies in Caenorhabditis elegans  show that RIC-3 is expressed in almost all neurons and in the body-wall muscles. In situ hybridization of mouse brain with an RIC-3 probe produces a low signal in most regions of the brain, with higher levels in cerebellum and some hippocampal areas. Northern blot analysis demonstrates RIC-3 expression in human brain and in some peripheral tissues . Detailed analysis of the distribution of RIC-3 expression is an important step towards understanding its biological function. Therefore a series of qPCR experiments were carried out to determine the expression profile of RIC-3 and RIC-3e mRNA transcripts in human tissues. A panel of cDNAs synthesized from mRNA samples derived from different human tissues, including several distinct brain regions, was used. The first set of primers (see Materials and methods section for details) was designed to amplify a product located within exon 6 of RIC-3. This set of primers recognizes all known transcripts except for the truncated RIC-3d form (GenBank® nucleotide sequence database accession numbers AY326436/AY358475, see Figure 2B). A second set of primers was designed to specifically amplify the new variant RIC-3e. Since exons 4 and 5 are missing in this variant, the reverse primer was designed across the junction of exon 3 with exon 6. As expected, a single product corresponding to RIC-3e was amplified from cDNAs from different tissues using this set of primers (result not shown). The results presented in Figures 3(A) and 3(B) indicate a tight correlation of the distribution and level of expression of the novel RIC-3e variant and the major RIC-3 isoform. The human RIC-3 gene is expressed predominantly in the central nervous system, with the highest levels detected in the cerebellum and pituitary gland. These results on the RIC-3 mRNA expression levels in different areas of human brain are in general accordance with recently published work which describes RIC-3 protein localization in the rat brain . In peripheral tissues, high levels of RIC-3 are observed in the pancreas and testis, with much lower levels of expression in other tissues.
Real-time qPCR expression analysis of
RIC- 3 variants and α 7- nAChR in a panel of human tissues
Since RIC-3e has a similar expression profile to RIC-3, the functional significance of this new variant remains elusive. However, despite the similarity in tissue distribution of the two RIC-3 variants, cell-type- or cell-compartment-specific variations in expression cannot be excluded. A detailed comparison of RIC-3 and RIC-3e proteins in tissues at the single-cell resolution level will be required to address this hypothesis.
RIC-3 was shown to be necessary for the functional expression of α7-nAChRs [13,14]. However, a detailed comparative expression analysis of both genes is not available. Therefore qPCR of α7-nAChR was performed on the same panel of human tissues as used for RIC-3 in order to determine if RIC-3 and α7-nAChR transcripts are coherently expressed (Figure 3C). Figure 3(C) shows that α7-nAChR transcripts are widely expressed within the central nervous system. The highest levels of α7-nAChR transcripts were found in the cerebral cortex, hippocampus and amygdala. This quantitative analysis correlates well with the distribution pattern of α7-nAChR described by in situ hybridization and [I125]Bgt-binding studies [20,21]. Analysis of the abundance of α7-nAChR transcripts in peripheral tissues shows a high level of expression in the human testis. Interestingly, some previous publications have suggested a role for α7-nAChR in the reproductive system [22,23]. Comparison of the distribution patterns of RIC-3 and α7-nAChR shows an overlapping profile of the two genes, which are both expressed at higher levels in the central nervous system than in the periphery. In general, all of the tissues that express α7-nAChR also express significant levels of RIC-3, consistent with the proposed function of RIC-3 as a chaperone of nicotinic receptors . Nevertheless, high levels of expression of RIC-3 are also seen in some brain regions (corpus callosum, pituitary gland and the cerebellum) which express relatively low levels of α7-nAChR. This suggests that RIC-3 might possess other, as yet unknown, functions which are not confined to the process of maturation of α7-nAChR.
Analysis of functional activity of RIC-3e
RIC-3 was shown to promote the folding, assembly and membrane targeting of recombinant α7-nAChR in different mammalian cell lines [13,18]. In particular, co-expression of RIC-3 and α7-nAChR resulted in the formation of surface α-Bgt-binding receptors in host cells that are otherwise unable to form functional α7-nAChRs, such as HEK-293, COS and tsA201 cells [13,17,18]. As the novel RIC-3 variant RIC-3e lacks the evolutionarily conserved coiled-coil domain and part of the C-terminal region present in RIC-3 proteins, we determined the capacity of RIC-3e to mediate proper membrane expression of α7-nAChRs in a cellular background which is non-permissive for functional expression of α7-nAChRs. HEK-293 cells were transfected with α7-nAChR, either alone or in combination with RIC-3 or RIC-3e. In order to detect α7-nAChR on the cell surface, live cells were incubated with fluorescently labelled α-Bgt. As expected, expression of human α7-nAChR alone did not result in the formation of α-Bgt-binding receptors (Figures 4A and 4B), On the contrary, co-expression of α7-nAChR with either RIC-3 (Figures 4C and 4D) or RIC-3e (Figures 4E and 4F) resulted in α-Bgt binding to cell membranes, indicating that both isoforms are able to promote surface expression of mature α7-nAChRs. Similar results were also observed using CHO-K1 cells (results not shown).
α-Bgt labelling of α7-nAChR in HEK-293 cells
In order to demonstrate that co-expression of RIC-3e not only leads to expression of α7-nAChRs on the plasma membrane, but also promotes the formation of functionally active α7-nAChR channels, we performed measurements of Ca2+ influx, as this channel is known to possess a high conductivity for Ca2+ ions in the activated state. HEK-293 cells were transiently transfected with constructs encoding human α7-nAChR together with RIC-3 or RIC-3e and with each plasmid alone as a control. Subsequently, cells were stimulated with nicotine in the absence or presence of the positive allosteric modulator PNU-120596 . The latter was necessary in order to increase the open-time of the α7-nAChR (which is known to desensitize very rapidly) such that the Ca2+ influx is sufficient to be measured using a FLIPR system. In fact, we have shown previously that potentiation of the activity of α7-nAChR with PNU-120596 was necessary in order to enable functional measurements of Ca2+ flux using a FLIPR system, depending on the cell line used .
As expected, treatment with nicotine alone did not elicit any measurable changes in the intensity of fluorescence (Figure 5). Conversely, the addition of nicotine after treatment with PNU-120596 resulted in a significant increase in intracellular Ca2+ concentrations. Importantly, this effect was only observed in cells which had been co-transfected with α7-nAChR and RIC-3 or α7-nAChR and RIC-3e. Moreover, the increase in fluorescence intensity was completely abolished when the cells were pretreated with MLA, a selective antagonist of α7-nAChR, thus confirming the specificity of the results obtained. In all experiments, the magnitude of the functional response of co-transfected α7-nAChR and RIC-3e was comparable with α7-nAChR in combination with RIC-3. More detailed studies of the two splice variants cannot be made in this system as subtle differences may be masked by the strong potentiation induced by PNU-120596. Taken together, we can conclude that RIC-3e is a functional splice variant which can play a role in the expression of functional α7-nAChRs at the plasma membrane. When compared with RIC-3, RIC-3e contains a deletion of the coiled-coil domain and part of the C-terminal domain. Deletion analyses of RIC-3 published previously suggested that ablation of the coiled-coil domain does not influence the capability of RIC-3 to modulate the expression of nicotinic and 5-HT3 receptors [17,25]. No changes were found in the chaperone activity of naturally occurring RIC-3 variants lacking the coiled-coil domain in Drosophila melanogaster . The results of the present study demonstrate that a variant, which does not contain the coiled-coil domain region (plus part of the adjacent C-terminal section) naturally exists and promotes α7-nAChR surface expression and functional receptor activity. Coiled-coil domains are known to be important for protein–protein interactions in the organization of molecular scaffolds, in addition to other functions . At least two other regions of RIC-3 have been predicted to be involved in protein–protein interactions  that may be sufficient to form the scaffold which recruits the complex necessary for the maturation of α7-nAChRs. The role of the missing segment may be to gather additional regulatory partners, to specify differential subunit composition of mature receptors or may be related to as yet undiscovered functions of RIC-3.
Determination of functional α7-nAChR activity using a FLIPR system
In summary, in the present study we report the existence of a new human variant of RIC-3 which lacks the coiled-coil domain, but is still capable of promoting the functional expression of α7-nAChRs in mammalian cells. The detailed analysis of the expression patterns of RIC-3, RIC-3e and α7-nAChRs has shown that both variants are very similarly distributed in human tissues, with a major overlap with α7-nAChR expression in the nervous system. Further studies addressing the biological function of RIC-3 and its variants are necessary to clarify the specific roles of the various isoforms and to identify new targets of this important regulator of neurotransmitter receptor expression.
threshold cycle value
fetal bovine serum
fluorescence imaging plate reader
- HEK-293 cell
human embryonic kidney cell
nicotinic acetylcholine receptor
resistant to inhibitors of cholinesterase-3
We thank Dr Giuseppe Pollio for proving support for cloning and molecular biology and Dr Hendrick Bothmann for helpful discussions regarding FLIPR experiments.
This work was supported by the Provincia di Siena [grant number 181843] awarded to T.F.
The nucleotide sequence data reported for the human RIC3e mRNA will appear in the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AM422214.