Keywords:

SYNOPSIS

The serotonin 5-HT 4 receptor is a member of the seven transmembrane-spanning G proteincoupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In this study, we isolated and characterized the 5'-flanking region of the human 5-HT 4 receptor (h5-HT 4 ). We demonstrate the existence of a novel exon which corresponds to the 5' untranslated region (UTR) of the h5-HT 4 receptor gene. RNase protection analysis and RT-PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT 4 receptor gene is located at -3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT 4 receptor was characterised. The promoter region lacks TATA and CAAT canonical motivefs in the appropriate location but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the [-3299/-3050] gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region [-220/-61] located in the long 5'-UTR. Mutational analysis, gel shift and transfection assays identified a Nkx2.5 like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT 4 receptor gene expression involving distinct promoters and non-coding exons.

INTRODUCTION

Serotonin (5-HT) is a major neurotransmitter both in the central nervous system and peripheral tissues. Of the 14 mammalian 5-HT receptor subtypes identified so far, all are Gprotein-coupled receptors (GPCRs) to the exception of the 5-HT 3 receptor that belongs to the superfamily of ligand-gated ion channels [START_REF] Hoyer | Molecular, pharmacological and functional diversity of 5-HT receptors[END_REF]. The 5-HT 4 receptor was first described as a potent 5-HT receptor able to induce cAMP production in primary neuronal cultures from mouse colliculi [START_REF] Dumuis | A nonclassical 5-hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system[END_REF]. Subsequent pharmacological studies have demonstrated the existence of 5-HT 4 receptor responses in the heart, adrenal gland, bladder and digestive tract of various species including human [START_REF] Langlois | 5-HT 4 receptor ligands: applications and new prospects[END_REF][START_REF] Hegde | Peripheral 5-HT4 receptors[END_REF]. In mammalian brain, activation of the 5 -HT 4 receptor increases memory and learning [START_REF] Marchetti | Differential modulation of the 5-HT(4) receptor agonists and antagonist on rat learning and memory[END_REF]. Accordingly, the 5-HT 4 receptor stimulates acetylcholine release in rat frontal cortex [START_REF] Maillet | New insights into 5-HT 4 receptor: a novel therapeutic target for Alzheimer's disease?[END_REF][START_REF] Lezoualc'h | The serotonin 5-HT 4 receptor and the amyloid precursor protein processing[END_REF] and regulates the processing of the amyloid precursor protein [START_REF] Robert | The Human Serotonin 5-HT4 Receptor Regulates Secretion of Non-amyloidogenic Precursor Protein[END_REF][START_REF] Maillet | Crosstalk between Rap1 and Rac regulates secretion of sAPPα[END_REF]. In peripheral tissues, the 5-HT 4 receptor has been shown to regulate gastrointestinal tract motility, intestinal electrolyte secretion and bladder contraction [START_REF] Hegde | Peripheral 5-HT4 receptors[END_REF].

In the heart, 5-HT 4 receptors exert strong positive chronotropic, inotropic and lusitropic effects in human and pig atria. However, unlike β-adrenergic receptors, the 5-HT 4 receptor exerts its effects exclusively on the atrial tissue and has no functional effects on ventricles [START_REF] Jahnel | Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle[END_REF][START_REF] Schoemaker | 5-Hydroxytryptamine stimulates human isolated atrium but not ventricle[END_REF]. In addition, the cardiac effects of the 5-HT 4 receptor are restricted to human and pig atria and are absent from a large number of laboratory animals, such as rat, guinea pig, rabbit and frog [START_REF] Kaumann | 5-HT4-like receptors in mammalian atria[END_REF][START_REF] Ouadid | Serotonin increases calcium current in human atrial myocytes via the newly described 5hydroxytryptamine4 receptors[END_REF]. Regarding 5-HT 4 receptor associated cardiac disorders, there are in vivo, in vitro, and clinical evidence that these receptors can trigger atrial arrhythmia [START_REF] Yusuf | 5 -hydroxytryptamine and atrial fibrillation: how significant is this piece in the puzzle?[END_REF]. Therefore, the regulation of 5-HT 4 receptor expression is of particular importance to understand their involvement in this cardiac disorder.

Several groups have isolated and sequenced C-terminal splice variants of the human 5-HT 4 (h5-HT 4 ) receptor. Eight C-terminal isoforms and one internal splice variant have been currently identified and named h5-HT 4(a) , h5-HT 4(b) , h5-HT 4(c) , h5-HT 4(d) , h5-HT 4(e) , h5-HT 4(f) , h5-HT 4(g) , h5-HT 4(n) and h5-HT 4(hb) [START_REF] Blondel | Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus[END_REF][START_REF] Claeysen | Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain[END_REF][START_REF] Bender | Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant[END_REF][START_REF] Mialet | Isolation of the serotoninergic 5-HT4(e) receptor from human heart and comparative analysis of its pharmacological profile in C6-glial and CHO cell lines[END_REF][START_REF] Vilaro | Cloning and characterization of a novel human 5-HT(4) receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT(4) receptor variants[END_REF]. These receptors are heptahelical transmembrane receptors that are positively coupled to adenylyl cyclase. The h5-HT 4 receptor gene is located on chromosome 5, bands 5q31-q33 [START_REF] Claeysen | Assignment of 5-hydroxytryptamine receptor (HTR4) to human chromosome 5 bands q31-q33 by in situ hybridization[END_REF] and contains 5 exons encoding the common transmembrane domains of the h5-HT 4 receptor as well as eight alternatively spliced cassettes which code for the internal and C-terminal splice variants [START_REF] Bender | Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant[END_REF]. The 5'-untranslated region (5'-UTR) of the h5-HT 4 receptor gene has recently been characterised from human placental cDNA. The 5'-UTR is extremely long in this tissue since it reached more than 5100 bp upstream from the translation start site and consists of 25 exons and a part of exon 26 which includes the first methionine (Figure 1A) [START_REF] Hiroi | Identification and characterization of the human serotonin-4 receptor gene promoter[END_REF]. Thus, according to this study the entire h5-HT 4 receptor gene spans more than 700 kb and is divided in at least 38 exons.

To date, there is no detailed information on the identification of the cis-acting elements which may be involved in the regulation of h5-HT 4 receptor expression. Therefore, to further understand the regulation of the h5-HT 4 receptor gene and its potential implication in cardiac disorders such as atrial arrhythmia, we characterised its 5'-UTR in human atrial RNA. RT-PCR experiments and RNA protection assay showed a major transcription start site located within intron 25 at -3185 bp relative to the first ATG codon. In addition to the promoter element, enhancer activity was found within intron 25 but in a region (-220/-61) located in the 5'-UTR. Mutational analysis, gel s hift and transfection assays identified a Nkx2.5 like binding site as a regulatory sequence of this enhancer.

MATERIALS AND METHODS

Surgery

All protocols for obtaining human tissue were approved by the ethics committee of GREBB institute (Hôpital de Bicêtre, Université de Paris-Sud). Specimens of right atrial appendages were obtained from patients undergoing heart surgery for coronary artery diseases or valve replacement at the Institut Hospitalier Jacques Cartier (Massy, France).

Cell culture and products

The human neuronal neuroblastoma cell line IMR32, human embryonic kidney (HEK) and human cervix epitheloid carcinoma (HeLa) cells were purchased from ATCC (Rockville, USA) and were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum (FCS), 1% non essential amino acids and antibiotics in humidified 5% CO 2 . COS-7 cells were cultured as previously described [START_REF] Blondel | Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus[END_REF]. Primary cultures of neonatal rat ventricular myocytes were prepared from hearts of 2-to 3-day-old Sprague-Dawley rat pups. Briefly, hearts were digested with collagenase and pancreatin.

Myocytes were then purified over a Percoll gradient and were plated at a density of 1 x 10 6 cells per 35-mm dish and cultured overnight in DMEM containing 5% FCS and 10% horse serum. All media, media supplements and sera were obtained from Life Technologies Inc. PCR Reactions were performed with High Fidelity Taq DNA polymerase (Roche Diagnostics, Meylan, France). The sequence of S124, S125, S137, S138 and β-actin primers have been previously described [START_REF] Blondel | Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus[END_REF][START_REF] Hiroi | Identification and characterization of the human serotonin-4 receptor gene promoter[END_REF].

Oligonucleotides

Reverse Transcription -Polymerase Chain Reaction (RT-PCR)

Total RNA was prepared from human atrial tissues and cell lines using the Trizol RNA purification system (Life Technologies Inc.). After purification, RNA was treated with DNase I (Life Technologies Inc.) and five µg of total RNA were then hybridized with oligo(dT) primer and reverse transcribed using Superscript reverse transcriptase II (Life Technologies Inc.). The resulting single strand cDNAs were used as templates in two successive PCR reactions using the different primers indicated in the text. Both PCR reactions were performed using the following cycle conditions: denaturation for 1 min at 94°C, annealing for 30 s at 58°C, and extension for 1 min 30 sec at 72°C with a final extension step for 8 min at 72°C.

The PCR products were electrophoresed on 1-2% agarose gel containing 0.01% ethidium bromide and photographed under U.V. irradiation.

RNase protection assay (RPA)

Total RNA from human atrium was analysed by RPA using the RPA III kit (Ambion, Austin, TX, USA) and an antisense RNA probe directed against the 5'-flanking sequence (-3362/-2970). Template DNA required for the synthesis of the RNA probe was obtained by PCR amplification of the BAC clone CTB-160O22 (Research Genetics, Huntsville, USA; GenBank accession no AC008627.7). The RNA probe was transcribed (Maxiscript T7 kit; Ambion) in presence of [α-32 P]UTP 800 Ci/mmol (Perkin Elmer Life Science products, France) and purified on a 5% polyacrylamide gel. The radiolabeled RNA probe was then hybridised with 50 µg of total RNA from human atrium or HEK cells at 42°C for 18 h. After digestion with RNase A and RNase T1, protected RNA fragments were separated on an 8 M urea/7% acrylamide gel. The gel was exposed to X-Omat AR film for 3 days (Eastman Kodak) at -80°C.

Reporter construct and luciferase assay

DNA fragments were constructed by PCR using the BAC clone CTB-160O22 (GenBank accession no AC008627.7) as a template and were cloned into the luciferase reporter vector, pGL3 basic in either KpnI/BglII or KpnI/XhoI restriction sites (Promega). The authenticity of the nucleotide sequences was confirmed by DNA sequencing. For cell lines, transient transfections were carried out in 24 well plates using polyethylenimine as previously described [START_REF] Boussif | A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine[END_REF]. Primary culture of rat neonatal cardiomyocytes maintained 2 days in vitro were transfected with lipofectamine (Life Technologies) according to the manufacturer's instructions. After transfection, myocytes were cultured overnight in DMEM with 5% horse serum, then in serum-free medium. After 24 h, cells were harvested and assayed for luciferase activity. Luciferase measurements were carried out using the Promega luciferase assay system according to manufacturer instructions in a Lumat luminometer. Results are expressed relative to the luminescence obtained with the promoter-less pGL3 basic vector. Each transfection experiment was done in quadruplicate, repeated at least 3 times and was normalized per mg of cell protein.

Site directed mutagenesis

Mutations in the

Electrophoretic mobility shift assay and identification of DNA-binding Proteins

Nuclear extracts were prepared from IMR32 cell line as previously described [START_REF] Schreiber | Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells[END_REF]. Nkx2.5

protein was prepared by coupled in vitro transcription/translation of a T7-driven Nkx2.5 plasmid in reticulocyte lysate by using a TNT kit (Promega). The Nkx2.5 gel retardation probe was constructed b y annealing complementary synthetic oligonucleotides (5'-AAACAAGTCATAATAGGAAGCA-3'). The oligonucleotide probe was end-labelled with γ-[ 32 P]ATP (3000 Ci/mmol) (Perkin Elmer Life Science products, France) and T4 polynucleotide kinase (Promega) and purified on a G-25 column. The following doublestranded oligonucleotides containing the mutated consensus sequences were used for competition assays: Nkx2.5 mut (5'-AAACAAGTCACGGTAGGAAGCA-3'). Ets-1 probe has already been described [START_REF] Hadri | Basal transcription of the mouse sarco(endo)plasmic reticulum Ca 2+ -ATPase type 3 gene in endothelial cells is controlled by Ets-1 and Sp1[END_REF]. Nuclear extracts (20 µg) or 3 µl of programmed lysate were incubated with 0.5 ng of [ 32 P] double-stranded probe in 20 µl of 20 mM HEPES pH 7.9, 100 mM NaCl, 20 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 % glycerol and 1 µg of poly(dI-dC) (Amersham Biosciences). For competition studies, before the addition of Nkx2.5 probe, nuclear extracts were preincubated for 10 min at room temperature with a 50-fold excess of unlabeled Nkx2.5 mut and Ets-1 oligonucleotides. Reactions mixtures were incubated for 30 min at room temperature and DNA-protein complexes were resolved on a 6% nondenaturing polyacrylamide gel at 20 mA for 3 hr in 0.5× TBE (45 mM Tris-borate and 1 mM EDTA).

Gels were vacuum-dried and were visualized using the Molecular Dynamics Storm PhosphorImager (Amersham Biosciences).

DNA affinity purification of nuclear proteins were performed using 300 µg of nuclear extracts

The 5'-biotinylated oligonucleotides MEF2-Control (AAGCTCGCTCTAAAAATAA-CCCTGTCCCTGGT), Mut-MEF2-Contro l (AAGCTCGCTCTAAGGCTAACCCTGTCCC-TGGT) and MEF2-5-HT 4 -R (TTAGTATTGTTTATTAAATATTTCCACAAAAC), were first coupled to streptavidine agarose beads (Sigma) for 1 hr at 4°C. Nuclear extracts were then incubated for 2 hrs with the precoated beads. Beads were washed three times and boiled in reducing sample buffer to elute the bound proteins. Proteins were separated on a 10 % polyacrylamide gel and electroblotted onto Amersham Pharmacia Biotech PVDF membrane.

Proteins were detected using anti-MEF-2 polyclonal antibody (Santa Cruz, H-300, 1:500) and visualized by the chemiluminescence system (Amersham Biosciences).

Statistical analysis

An unpaired Student´s t-test was used to evaluate differences between means; differences were considered significant when p < 0.05.

RESULTS

Isolation of the 5' end of the h5-HT 4 receptor cDNA

To further identify the 5' end of the h5-HT 4 receptor cDNA in human atria, we carried out successive RT-PCR experiments on total RNA extracted from human atria using various oligonucleotide primers based on the h5-HT 4 receptor genomic DNA sequence (Genbank accession no AC008627.7). Because h5-HT 4 receptor mRNA are weakly expressed in their different target tissues, especially in the atrium [START_REF] Blondel | Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus[END_REF][START_REF] Medhurst | Quantitative mRNA analysis of five C-terminal splice variants of the human 5-HT(4) receptor in the central nervous system by TaqMan real time RT-PCR[END_REF], we performed nested PCR to amplify the 5' upstream sequence. To prevent any amplification of genomic DNA, sense and antisense primers were respectively selected on intron 25 and exon 27 (Figure 1A). predicted products (Figure 2B). In contrast, no PCR product was synthesized from reverse transcribed RNA with couple of primers [-3299/-2166] followed by [-2837/-2318] although an obvious fragment of the expected size was amplified from genomic DNA (Figure 2B). The specificity of the PCR products was confirmed by Southern blot using the 32 P-5'-end-labeled internal -2392 primer (Figure 2B, right panel). Altogether these results indicated that the transcription start site of the h5-HT 4 receptor gene in human atria was located within a region between -3299 bp and -2837 bp from the ATG codon.

Identification of the transcription start site of the h5-HT 4 receptor gene

To delineate the region containing the transcription start site, PCRs were performed using 3'

antisense primers adjacent to primer -2340 and progressively further 5' sense primers (Figure 3). cDNA from human atria and human genomic DNA were compared. The nested PCR products obtained from this analysis are shown in Figure 3A. 3A, right panel). To map the transcription start site with another approach, we performed RPA analysis with total RNA isolated from human atrial tissues or HEK cells using a 32 P-labelled RNA probe encompassing the sequence -3362/-2970 of the h5-HT 4 receptor gene. As shown in Figure 3B (lane 1), the 392 bp probe hybridised with human atrial RNA was partially protected against degradation and gave a 200 bp fragment. This result demonstrated the presence of one major start site within the 5-HT 4 receptor gene in human atria around position -3185. The absence of any specific signal in the lane corresponding to the RNA extracted from HEK cells is in accordance with the observation that these cells do not express the h5-HT 4 receptor as assessed by RT-PCR (Figure 3B). Altogether these results

indicate that the main transcriptional start site of the h5-HT 4 receptor gene resides between -3192 and -3134 bp in human atria.

Next, to check whether this transcriptional start site was unique to the heart, we carried out nested RT-PCR experiments on RNA extracted from human placenta (Figure 3C). In this tissue the major transcriptional start site of the h5-HT 4 receptor has been previously located in exon 1 which is at more than 450 kb from the ATG codon [START_REF] Hiroi | Identification and characterization of the human serotonin-4 receptor gene promoter[END_REF]. As shown in Figure 3C, no PCR product was obtained in human placenta with primers [-2837/-2166] followed by [-2762/-2318] whereas a fragment of 444 bp was amplified from human atrial cDNA.

Similarly, the primer pair [-3093/-2392] allowed the amplification of a DNA fragment of 700 bp from the human heart cDNA product amplified with the primer pair [-3134/-2318] (Figure 3A). No PCR product was observed when the same experiment was performed on cDNA of human placenta (data not shown). We also demonstrated the presence in heart and placenta tissues of cDNA corresponding to the constitutively expressed β-actin gene (Figure 3C), as well as the absence of actin PCR product in a control without reverse transcriptase (Figure 3C). Therefore, signals obtained in our study were not due to any contaminating genomic DNA since no bands were observed when RNA was directly amplified. Altogether, these data indicate that the long 5' UTR and the -3185 bp start site are unique to the heart. With respect to the finding of Hiroi et al. [START_REF] Hiroi | Identification and characterization of the human serotonin-4 receptor gene promoter[END_REF], we named this new long exon which contains the 5' UTR located on intron 25 and exon 26, exon 1 bis (Figure 3D, upper panel).

Interestingly, we found that the start site present in human placenta appeared also to be used in human atria since we were able to detect by PCR a 220 bp product which corresponds to a DNA fragment lying between exon 1 and exon 4 (Figure 3D, lower panel). This nested PCR was performed using primers [S137/S124] and [S138/S125] which have been previously

shown to specifically amplify these exons [START_REF] Hiroi | Identification and characterization of the human serotonin-4 receptor gene promoter[END_REF]. These results suggest that the h5-HT 4 receptor gene may use several transcription initiation sites (Figure 3D, upper panel).

Transcriptional activity of the h5-HT 4 receptor promoter

The genomic 5' flanking sequence lying between position -4298 and -3100 did not contain any canonical TATA sequence nor a CAAT box (Figure 1B). Nevertheless, to test whether the region located upstream of exon 1 bis behaved as a functional promoter, three 5' deletion fragments were generated by PCR and subcloned into the promoter-less vector pGL3-Basic (Figure 4). The largest DNA fragment corresponded to sequence -4298/-3050 while the others corresponded to shorter proximal sequences extending from -3639 to -3050 and -3299 to -3050 (Figure 4). To date, there is no in vitro cardiac cellular system expressing endogenous 5 -HT 4 receptors which could allow such a functional study. Therefore, the promoter activities of the luciferase constructs were evaluated by transient transfection in three human cell lines, IMR32, HeLa and HEK cells as well as in monkey COS-7 cells.

Among these cell lines, only human neuroblastoma IMR32 cells express 5-HT 4 receptor mRNA ( [START_REF] Robert | The Human Serotonin 5-HT4 Receptor Regulates Secretion of Non-amyloidogenic Precursor Protein[END_REF], and data not shown). After transfection in COS-7 cells, the different reporter constructs did not produce any significant increase in luciferase activity compared to the control pGL3-basic vector (Figure 4) although a strong signal was detected upon transfection of the cytomegalovirus (CMV) promoter linked to the luciferase gene (data not shown). In contrast, all three constructs activate luciferase expression to approximately 1.5-to 7-fold above control value in transiently transfected human HeLa, HEK and IMR32 cells (Figure 4).

The highest relative luciferase expression (up to 8 fold the control value with -3299/-3050 construct) was observed in HeLa cells (Figure 4). The sequential 5' deletions of the region from -4298 to -3299 significantly increased promoter activity in the three human cell lines suggesting the presence of repressor elements in this promoter domain (Figure 4).

Transcriptional activity of exon 1 bis of the h5-HT 4 receptor

The h5-HT 4 receptor comprised more than 3.1 kb in its 5' UTR located in exon 1 bis. Since transcriptional regulation by exons has been reported for several genes [START_REF] Boukouvala | Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine Nacetyltransferase type 2 in mice[END_REF][START_REF] Stickeler | The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4[END_REF], we examined the promoter activity of the long 5' UTR of the h5-HT 4 receptor. To this end, we generated by PCR a series of constructs containing serial 3' deletions of exon 1 bis which were sub-cloned into the pGL3-Basic vector (Figure 5). The constructs were then transiently transfected into HeLa, HEK, IMR32 and COS-7 cells and assayed for luciferase activity (Figure 5). As observed with previous constructs, no luciferase activity was detected in COS-7 cells with any of these new constructs (Figure 5). A modest, not significant increase in luciferase activity was obtained with the two constructs, -2340/-61 and -1200/-61 when transfected in human cell lines (Figure 5). Deletion of the region -1200 to -845 led to a significant enhancement in transcriptional activity in human cell lines as compared with the pGL3-basic vector (Figure 5). Most importantly, deletion from -845 to -220 produced a greater than 10-fold increase in luciferase activiy in IMR32 and HEK cells (Figure 5). Again, the highest relative luciferase activity was observed in HeLa cells (more than 40 fold the control value) (Figure 5).

An Nkx2.5 like element is required for optimal transcriptional activity of the [-220/-61] region

The prediction of potential regulatory elements within the [-220/-61] region was determined by using MatInspector [START_REF] Quandt | MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data[END_REF] and the TFSearch [START_REF] Heinemeyer | Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL[END_REF]. We found that it contains putative binding Finally, to test the potential involvement of the transcription factor Nkx2.5 in the regulation of the transcriptional activity of the [-220/-61] region in cardiac cells, we carried out co-transfection experiments in primary neonatal rat ventricular myocytes. Figure 6D shows that transfection of an expression vector encoding Nkx2.5 enhanced the transcriptional activity of the [-220/-61] region in primary neonatal cardiomyocytes. These data indicate that Nkx2.5 may participate in the regulation of 5-HT 4 receptor gene expression.

DISCUSSION

In the present study we report the functional characterization of the 5'-flanking region of the h5-HT 4 receptor. RT-PCR experiments combined with the sequencing of the 5'-flanking region of the h5-HT 4 receptor showed that the long 5'-UTR (3.1 kb) lays between the transcription initiation site and the first ATG. Such long 5'-UTR may reduce RNA translation and lead to low levels of expressed transcripts [START_REF] Meijer | Control of eukaryotic protein synthesis by upstream open reading frames in the 5'-untranslated region of an mRNA[END_REF] as it is the case for the h5-HT 4 receptor in the heart [START_REF] Medhurst | Quantitative mRNA analysis of five C-terminal splice variants of the human 5-HT(4) receptor in the central nervous system by TaqMan real time RT-PCR[END_REF][START_REF] Kaumann | Comparison of the densities of 5-HT4 receptors, beta 1 -and beta 2 -adrenoceptors in human atrium: functional implications[END_REF]. Indeed, self-complementary sequences can form stable stem-loop structures that interfere with the assembly of the preinitiation complex. These stem loops can be further stabilized by the interaction with RNA-binding proteins. Furthermore, the presence of ATG codons located upstream of the start site can inhibit translation by causing premature initiation and thereby preventing the ribosome from reaching the ATG start codon [START_REF] Cazzola | Translational pathophysiology: a novel molecular mechanism of human disease[END_REF].

Sequence analysis of the 5'-untranslated flanking region of the h5-HT 4 receptor gene indicated that the region upstream of the putative transcriptional start site does not contain any of the typical characteristics of promoter regions such as a TATA box, CAAT box, or the GCrich region. The TATA-less promoter is characteristic of GPCRs and examples include promoters of the serotonin 5-HT 2A and 5-HT 2C receptors [START_REF] Zhu | Characterization of the human 5-HT2A receptor gene promoter[END_REF][START_REF] Xie | The human serotonin 5-HT 2C[END_REF]. Interestingly, inspection of the sequence around the transcription initiation site revealed a consensus sequence for an initiator element (INR) which consists of the sequence TTCACTTT (Figure 1B) [START_REF] Smale | Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes[END_REF]. The INR is known to be functionally analogous to the TATA box since it determines the site of transcription and its direction [START_REF] Smale | Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes[END_REF].

In our study, we found that, in human atrium, the long 5'-UTR of the h5-HT 4 receptor mRNA is located -3185 bp upstream of the translation initiation site and is encoded by a single exon (exon 1 bis). This start site is unique to the heart and not present in placenta.

However, the start site previously described in placenta was also detected in the heart (Figure 3B) suggesting that the h5-HT 4 receptor gene may use multiple transcription initiation sites. This is in accordance with previous studies showing that 5-HT 1A , 5-HT 2A and 5-HT 2C receptor genes have several transcription start sites [START_REF] Zhu | Characterization of the human 5-HT2A receptor gene promoter[END_REF][START_REF] Parks | The serotonin 1A receptor gene contains a TATA-less promoter that responds to MAZ and Sp1[END_REF][START_REF] Xie | The human serotonin 5-HT 2C[END_REF]. Therefore, the 5'-UTR of the h5-HT 4

receptor is a result of the use of the two alternate promoters. It does not change the proteincoding region, but may affect the transcription efficiency as well as the spatial and temporal expression of the h5-HT 4 receptor. The reason for the complex arrangement of numerous start sites spread over an extended domain remains obscure. Perhaps these mRNAs are differentially regulated at the level of translation in different cell types. Alternatively, the transcription initiation sites may be regulated independently, providing a potential mechanism to differentially control transcription of the gene in different cell types and tissues regions as it has been shown for several genes [START_REF] Chiu | Multiple controlling mechanisms of FGF1 gene expression through multiple tissue-specific promoters[END_REF].

Transient transfection assays with a series of the h5-HT 4 receptor promoter-luciferase constructs identified a ~1.2 kb fragment of 5'-nontranscribed sequence that was active as a promoter in the human cell lines, IMR32, HEK and HeLa and silent in COS-7 cells. These data suggest that this DNA fragment is necessary to drive cell specific expression of the 5- Nkx2.5 (also called Csx) is a member of the NK class of homeodomain proteins required for cardiac commitment, differentiation and morphogenesis [START_REF] Schwartz | Building the heart piece by piece: modularity of cis-elements regulating Nkx2-5 transcription[END_REF]. This transcription factor is continually expressed from embryonic stage to adulthood in human heart [START_REF] Shiojima | Molecular cloning and characterization of human cardiac homeobox gene CSX1[END_REF]. Nkx2.5 has been shown to regulate transcription of markers of cardiac hypertrophy such as the skeletal αactin [START_REF] Chen | Identification of novel DNA binding targets and regulatory domains of a murine tinman homeodomain factor, Nkx-2.5[END_REF] and the atrial natriuretic factor [START_REF] Durocher | The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium[END_REF]. However, Nkx2.5 by itself is a weak transactivator and requires additional interactions with other transcriptions factors such as serum response factor or GATA-4 [START_REF] Sepulveda | GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA binding targets: role for regulating early cardiac gene expression[END_REF][START_REF] Sepulveda | Combinatorial expression of GATA4, Nkx2-5, and serum response factor directs early cardiac gene activity[END_REF]. The potential MEF-2 binding site located in the proximity of the Nkx2.5 binding site would have been a good partner since MEF-2 response elements have been reported in several cardiac promoters and their mutations have been shown to decrease promoter activity [34,[START_REF] Di Lisi | Combinatorial cis-acting elements control tissue-specific activation of the cardiac troponin I gene in vitro and in vivo[END_REF]. However, when we performed DNA affinity purification using the MEF-2 like binding site as a probe, we were not able to detect any binding of MEF-2 proteins to this DNA sequence (data not shown). Thus the partners of NKx2.5 have to be defined.

In summary, we have identified a second transcription start site of the h5-HT 4 receptor gene which is under the control of a promoter region located at more than 3 kb from the first ATG codon. We also provide evidence that the long 5'UTR isolated in our study is only coded by one exon in human atrium. The [-220/-61] region contains a Nkx2.5 like element which regulates the transcriptional activity of the h5-HT 4 receptor gene.

  Numbering is relative to the first ATG codon. Primers used for the different experiments were designed based on the sequences of the 5-HT 4 receptor cDNA (GenBank accession no Y12507) and the BAC clone CTB-160O22 (GenBank accession no AC008627.7): Sense primers: -2106 (position -2106/-2084); -2340 (position -2340/-2318); -2711 (position -2711/-2689); -2762 (position -2762/-2740); -2837 (position -2837/-2815); -2994 (position -2994/-2972); -3093 (position -3093/-3071); -3134 (position -3134/-3112); -3192 (position -3192/-3170); -3299 (position -3299/-3277); antisense primers: +40 (position +40/+20); +131 (position +131/+113); -2166 (position -2166/-2188); -2318 (position -2318/-2341); -2392 (position -2392/-2415); -2970 (position -2970/-2947).

  Oct-1, MEF-2 and Nkx2.5 consensus binding sites within region [-220/-61] were introduced using QuickChange TM site-directed mutagenesis kit (Statagene) using the pGL3[-220/-61] construct as a template. The sequences of the mutated oligonucleotides used

  Using the sense/antisense primers [-2340/+131] and [-2106/ +40] in consecutive PCR reactions, a 2 kb DNA fragment was amplified (data not shown). It displayed full homology with the h5-HT 4 receptor genomic DNA sequence (Genbank accession no AC008627.7) indicating that the 5' UTR of the h5-HT 4 receptor in human atria extended at least upstream of position -2340 bp from the ATG codon (data not shown). The 5' UTR was further explored by carrying out nested PCR on cDNA using sense primers located upstream position -2340 (Figure 2A). Using as templates the products of a first PCR performed with the couple of primers [-2837/-2166], a second nested PCR performed with the sense/antisense primers [-2762/-2318] led to the amplification of products of expected sizes (Figure 2B). Similarly, two PCR reactions performed consecutively with couple [-2762/-2166] and couple [-2711/-2318] amplified the

  sites for the transcription factors Oct-1, MEF-2 and Nkx2.5. Therefore, we examined whether mutations in the binding sites of these transcription factors affected the enhancer activity of the [-220/-61] region. Mutations of the Oct-1 consensus binding site did not significantly influence the transcriptional activity of the [ -220/-61] region in IMR32 cells (Fig 6A) although these mutations have been previously shown to be effective in the loss of Oct-1 binding [43]. A mutation in the MEF-2 like binding site induced a slight decrease of the transcription activity (30%) (Fig 6A). However, we were unable to show any binding of MEF-2 proteins to this DNA sequence when we performed DNA affinity purification of nuclear proteins extracted from IMR32 cells (data not shown). Mutations in the Nkx2.5 like binding site reduced the enhancer activity of the [-220/-61] region by 70% (Fig 6A). These results indicate that the Nkx2.5 like consensus sequence plays a major role in the transcriptional activity of the [-220/-61] region.Gel mobility shift assay was conducted to determine whether the Nkx2.5 consensus sequence was able to bind nuclear proteins. Two major complexes were formed with the radio-labelled Nkx2.5 probe and proteins extracted from IMR32 cells (Figure6B, lane 1). All bound proteins can be displaced by 50-fold excess unlabeled Nkx2.5 consensus sequence (Figure6B, lane 3). Competition with a mutant Nkx2.5 binding site failed to completely abolish complex formation (Figure6B, lane 2) as did an excess of unlabelled consensus Ets-1 oligonucleotide (Figure6B, lane 4). To confirm that Nkx2.5 protein binds to the Nkx2.5 consensus sequence located in the [-220/-61] region, an electrophoretic mobility shift assay was performed with in vitro translated Nkx2.5 protein and Nkx2.5 wild type (WT) and mutated (Mut) probes. As shown in Figure6C, Nkx2.5 protein was bound to the WT probe and to a much lesser extent to the Mut probe . These results demonstrated that Nkx2.5 protein specifically binds to this Nkx2.5 like sequence.

HT 4

 4 promoter. Progressive deletion of the h5-HT 4 promoter resulted in an increase in luciferase activity in human cells but not in monkey COS-7 cells. This indicates that the sequences immediately upstream of the transcription start site probably contain positive elements that are required to enhance transcription in the human cell lines tested. Surprisingly, we found that the DNA region -845/-61 of exon 1 bis increased luciferase activity upon transfection in human cells. Deletion of the region located between -845 and -220 further enhanced luciferase expression indicating the presence of strong positive elements between position -220 and -61. Analysis of the [-220, -61] region by MatInspector and TransFac programs revealed that it contains putative binding sites for several transcription factors such as Oct-1 and Nkx2.5. Mutational analysis of either consensus sequences showed that the Nkx2.5 like binding site strongly regulates transcriptional activity of the [-220/-61] region. EMSA demonstrates specific binding of nuclear proteins to a portion of this region containing the Nkx2.5 binding site.

Figure 1 .

 1 Figure 1. A) 5' gene organization of the h5-HT 4 receptor in human atria. An arrow indicates the ATG translation initiation codon. B) Nucleotide sequence (GenBank accession no AC008627.7) of the 5' upstream flanking region of the h5-HT 4 receptor gene showing the position of various putative transcription factor binding sites (underlined). Bases relative to the ATG translation initiation codon (in boldface, designated +1) are numbered on the left and on the right. The intron sequence is shown in small letters and the coding sequence in bold letters.

Figure 2 .

 2 Figure 2. Isolation of a long 5' UTR in the 5-HT 4 receptor gene in human atria. A) Upper Panel. A schematic diagram representing the positions of sense (dark arrows) and antisense (grey arrows) primers on intron 25 used for RT-PCR experiments. The open arrow indicates the position of the probe used for the Southern blot. B) Results of agarose gel electrophoresis of nested PCR products are shown on the left panel. Molecular size markers are indicated in bp. Primer pairs used for the nested PCR are represented into brackets. The specificity of the PCR products were analysed by Southern blotting using the 32 P-5'-end-labeled internal -2392 primer. A 6 h exposure of the autoradiogram is shown on the right panel. gDNA; human genomic DNA.

Figure 3 .

 3 Figure 3. Identification of the region of the transcription start site of the 5-HT 4 receptor gene. A) Results of agarose gel electrophoresis of cDNA and genomic DNA (gDNA) of nested PCR products are shown on the left and right lower panels, respectively. Molecular size markers indicated in bp were run with the PCR products. The positions of primers are shown in the upper panel. Sense and antisense primers are in dark and grey arrows respectively. CT, PCR performed with water instead of cDNA is a negative control. B) RNase protection analysis of the h5-HT 4 receptor gene. The labelled RNA probe [-3362, -2970] was hybridised to 60 µg of human atrium RNA (lanes 1), 60 µg of HEK cells RNA (lanes 2), 60 µg of tRNA (lanes 3), 60 µg of tRNA without RNase A (lanes 4). The RNA molecular weight in bp is shown in lane 5. The position of a major protected band and its size is indicated by an arrows on the left. Asterisks show non-specific background. C) Agarose gel electrophoresis of PCR products showing that the -3185 bp start site is unique to the heart. The PCR experiment was performed on cDNA of human placenta and atrium using primers [ -2837/-2166] followed by [-2762/-2318]. These primers are specific of Exon 1A. Positive control was

Figure 4 .

 4 Figure 4. Transcriptional activity of the h5-HT 4 receptor promoter. The upper panel shows a diagram of the 5' flanking region of the h5-HT 4 receptor gene containing the large exon 1 bis encoding the long 5' UTR (-3185, +26). The schematic diagram on the lower left represents each deletion construct of the h5-HT 4 receptor promoter linked to the luciferase gene which were transfected into different cell lines (right panel). Results are expressed as relative luciferase activity expressed as fold expression over the promoter-less pGL3 Basic vector. Values reported in the figure represent the mean ± S.E.M of four independent transfections performed in triplicates. Bars bearing "*" (p<0.05), "**" (p<0.01), "***"(p<0.001) are statistically different from the control pGL3 Basic vector.

Figure 5 .

 5 Figure 5. Functional analysis of the 5' UTR of the h5-HT 4 receptor gene in different cell lines. 5' deletion construct of exon 1 bis were fused to the pGL3 Basic vector and transfected into the indicated cell lines. Schematic representation of the constructs tested and the relative luciferase activity are indicated on the left and right panel, respectively. Results are expressed as relative luciferase activity expressed as fold expression over the promoter-less pGL3 Basic vector. Values reported in the figure represent the mean ± S.E.M of four independent transfections. Bars bearing "**" (p<0.01), "***"(p<0.001) are statistically different from the control pGL3 Basic vector.

Figure 6 .Figure 1

 61 Figure 6. Identification of a Nkx2.5 like binding site that regulated the transcriptional activity of the [-220/-61] region. A) Effects of mutations in the potential MEF-2, Nkx2.5 and Oct-1 binding sites of the [-220/-61] region in exon 1 bis. Wild type (WT) and mutant constructs of the [-220/-61] region were transfected into IMR32 cells and luciferase activity was then assayed. Results are expressed as a percentage of the wild type pGL3[-220/-61] luciferase activity. Values represent the mean ± S.E.M of three independent transfections.

ACKNOWLEDGMENTS

We wish to thank Dr. Patrick Donzeau-Gouge, Institut Hospitalier Jacques Cartier, Service de Chirurgie Cardiaque, F-91349 Massy Cedex, France, for providing human atrial tissues. We are grateful to Dr. Izumo, Department of Medicine, Harvard Medical School, Boston, USA for providing expression vector encoding Nkx2.5


Rodolphe Fischmeister

H Criss Hartzell
email: criss.hartzell@emory.edu

chloride channels

Keywords: chloride channels, electrophysiology, cell volume, ion channel

Bestrophins are a newly-identified family of Cl channels. Mutations in the founding member of the family, human bestrophin-1 (hBest1), are responsible for a form of early-onset macular degeneration called Best vitelliform macular dystrophy. The link between dysfunction of hBest1 and macular degeneration remains unknown. Because RPE cells may be subjected to varying osmotic pressure due to light-dependent changes in the ionic composition of the sub-retinal space and because RPE cells may undergo large volume changes during phagocytosis of shed photoreceptor discs, we investigated whether bestrophin currents were affected by cell volume. When hBest1 and mBest2 were overexpressed in HEK 293, HeLa, and ARPE-19 cells, a new Ca-activated Cl current appeared. This current was very sensitive to cell volume. A 20% increase in extracellular osmolarity caused cell shrinkage and a ~70 -80 % reduction in bestrophin current. Decreases in extracellular osmolarity increased the bestrophin currents slightly, but this was difficult to quantify due to simultaneous activation of endogenous VRAC current. To determine whether a similar current was present in mouse RPE cells, the effect of hyperosmotic solutions on isolated mouse RPE cells was examined. Mouse RPE cells exhibited an endogenous Cl current that resembled the expressed hBest1 in that it was decreased by hypertonic solution. We conclude that bestrophins are volume sensitive and that they could play a novel role in cell volume regulation of RPE cells.

INTRODUCTION

Best vitelliform macular dystrophy (VMD2) is a degeneration of the macular region of the retina [START_REF] Petrukhin | Identification of the gene responsible for Best macular dystrophy[END_REF]Bakall et al., 1999;[START_REF] Caldwell | Bestrophin gene mutations in patients with Best vitelliform macular dystrophy[END_REF][START_REF] White | VMD2 mutations in vitelliform macular dystrophy (Best disease) and other maculopathies[END_REF]. Best disease often develops in childhood and is associated with the accumulation of a yellow fluid between the retina and the retinal pigmented epithelium (RPE) [START_REF] Gass | Stereoscopic atlas of macular diseases: Diagnosis and treatment[END_REF][START_REF] O'gorman | Histopathologic findings in Best's vitelliform macular dystrophy[END_REF]. The fluid accumulation in Best disease is consistent with abnormal fluid transport, which often involves Cl -channels. Recently, bestrophin, the protein product of the VMD2 gene, has been identified as a Ca 2+ -activated Cl -channel [START_REF] Sun | The vitelliform macular dystrophy protein defines a new family of chloride channels[END_REF][START_REF] Tsunenari | Structure-function analysis of the bestrophin family of anion channels[END_REF][START_REF] Qu | Two bestrophins cloned from Xenopus laevis Oocytes express Ca-activated Cl currents[END_REF]2004). Several mutants of human bestrophin-1 (hBest1), an isoform selectively expressed in the basolateral membrane of RPE [START_REF] Marmorstein | Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium[END_REF][START_REF] Bakall | Expression and localization of bestrophin during normal mouse development[END_REF], are responsible for Best disease and dominantly inhibit the Cl -conductance associated with wild type hBest-1 [START_REF] Qu | Two bestrophins cloned from Xenopus laevis Oocytes express Ca-activated Cl currents[END_REF][START_REF] Tsunenari | Structure-function analysis of the bestrophin family of anion channels[END_REF].

The connection between defective hBest1 function and the accumulation of lipofuscin-like material within and beneath the RPE is unknown. However, important roles of the RPE are to sustain photoreceptor renewal by phagocytosis of shed photoreceptor outer segments [START_REF] Lavail | Outer segment disc shedding and phagocytosis in the outer retina[END_REF] and to control the volume and composition of the sub-retinal space [START_REF] Gallemore | Retinal pigment epithelial transport mechanisms and their contributions to the electroretinogram[END_REF]. Both of these functions might be expected to produce changes in cell volume. RPE preparations have been shown to transport water from the retina to choroid [START_REF] Hughes | Effects of cyclic AMP on fluid absorption and ion transport across frog retinal pigment epithelium. Measurements in the open-circuit state[END_REF][START_REF] Gass | Stereoscopic atlas of macular diseases: Diagnosis and treatment[END_REF][START_REF] Cour | Osmotic properties of the frog retinal pigment epithelium[END_REF] and to support the vectorial traffic of ions and fluid (for a review, see [START_REF] Gallemore | Retinal pigment epithelial transport mechanisms and their contributions to the electroretinogram[END_REF]. Besides, RPE cells possess the appropriate ion channel and transport armamentarium to respond to hyposmotic swelling or hyperosmotic shrinkage by regulatory volume changes [START_REF] Cour | Osmotic properties of the frog retinal pigment epithelium[END_REF][START_REF] Kennedy | Volume regulation in cultured cells derived from human retinal pigment epithelium[END_REF]Civian et al., 1994;[START_REF] Adorante | Regulatory volume decrease in frog retinal pigment epithelium[END_REF]). Therefore, it is tempting to speculate that part of the RPE dysfunction in Best disease might involve abnormal RPE cell volume regulation, or abnormal Cl -channel responses to cell volume changes due to defective hBest1 Cl - channels. Interestingly, in diabetic retinopathy, another pathology with RPE transport defects, the regulatory cell volume response of RPE cells to shrinkage was found to be inhibited [START_REF] Civan | Prolonged incubation with elevated glucose inhibits the regulatory response to shrinkage of cultured human retinal pigment epithelial cells[END_REF]. In this study, we found that hyperosmotic shrinkage induced a profound inhibition of the bestrophin Cl -currents. In isolated mouse RPE cells, hyperosmotic shrinkage also suppressed part of the total Cl -current, which might indicate a contribution of bestrophins to the native RPE Cl -conductance.

METHODS

Heterologous expression of hBest1 and mBest2 in mammalian cell lines

cDNA clones of human Bestrophin-1 (hBest1) (GENBANK NM_004183, generously provided by Dr. Jeremy Nathans, Johns Hopkins University,), mouse Bestrophin-2 (mBest2) (ATCC, IMAGE clone ID: 4989959, GENBANK AY450428) or human cystic fibrosis transmembrane conductance regulator (hCFTR) (GENBANK M28668, provided by Dr. David Gadsby, Rockefeller University) were sequenced to verify that their sequence agreed with the published EMBL/GenBank/DDBJ sequences.

Cell lines used include HEK 293 (human embryonic kidney), ARPE-19 (human retinal pigment epithelium), and HeLa (human fibroblast). All cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). hBest1, mBest2 or hCFTR were transfected into these cells using Fugene-6 transfection reagent (Roche, Indianapolis, IN). Usually, 0.1 µg mBest2, 0.9 µg hBest1 or 0.1 μg CFTR cDNA was used to transfect one 35 mm culture dish. pEGFP (Invitrogen, Carlsbad, CA) was also transfected to identify transfected cells. The pEGFP plasmid alone (1 μg) was used as a transfection control. One day after transfection, cells were dissociated and replated on glass coverslips for electrophysiological recording. Transfected cells were identified by EGFP fluorescence and used for patch clamp experiments within three days after transfection.

Mouse retinal pigment epithelial cell isolation

All procedures with animals were designed to minimize pain and suffering and conformed to NIH guidelines. The animal protocols were approved by the Emory University Institutional Animal Use and Care Committee. Six to 12 week female mice were anesthetized using isoflurane (Baxter, Deerfield, IL) and decapitated. Eyes were removed and dissected under a microscope in a dissection plate containing a Ringer solution. The anterior segment and vitreous were removed by a circumferential incision at the limbus. The neural retina was gently peeled to obtain clean eye cups with the retinal pigment epithelial (RPE) layer exposed. The eye cups were then transferred into clean Ringer solution, washed gently, and then placed in 10 ml of isolation solution containing 2 mg papain (Sigma-Aldrich, St. Louis, MO) at 37 o C for 30 min, with a gentle agitation every 5 min. At the end of this 30 min period, the eye cups were transferred into a microfuge tube containing 1 ml Ringer, and RPE cells were dispersed by few taps on the tube. The cell suspension was plated on glass coverslips in culture medium and kept at 37oC for 2-10 hours prior to electrophysiological recordings.

Electrophysiological Recordings

Recordings were performed using the whole-cell recording configuration of the patch-clamp technique. Patch pipets were made of borosilicate glass (Sutter Instrument Co., Novato, CA), pulled by a Sutter P-2000 puller (Sutter Instrument Co.), and firepolished. Patch pipets had resistances of 1.5-3 MΩ when filled with the standard intracellular solution (see below). The bath was grounded via a 3-M KCl agar bridge connected to a Ag/AgCl reference electrode. Solution changes were performed by perfusing the 1-ml chamber at a speed of ~4 ml/min. To measure the steady-state currentvoltage relationship, the cells were voltage clamped from a holding potential of 0 mV with 750-ms duration pulses from -100 mV to +100 mV in 20-mV increments at 0.25 Hz.

Because the currents were usually time independent, most experiments used a 1350-ms duration voltage ramp from -100 to +100 mV delivered every 8 sec. The ramp was preceded with a 500 ms pulse to -100 and followed by a 500 ms pulse to +100 mV. Data were acquired by an Axopatch 200B amplifier controlled by Clampex 8.2 via a Digidata 1322A data acquisition system (Axon Instruments, Inc., Foster City, CA). Experiments were conducted at room temperature (20-24 o C). Liquid junction potentials were corrected using the liquid junction potential calculator in Clampex 8.2. Rectification ratios (RR) were determined from the quotient of the current measured at +100 mV divided by the current measured at -100 mV.

A question that arises in these studies is how the whole-cell patch clamp configuration influences cell volume changes. This situation was addressed by [START_REF] Ross | Membrane chloride conductance and capacitance in Jurkat T lymphocytes during osmotic swelling[END_REF] who predicted that whole-cell patch-clamped cells will swell indefinitely when exposed to the "infinite" hyperosmotic perfusion volume of the pipette. When extracellular osmolality is decreased (like in 0.9T), water starts entering the cell causing swelling. This raises the water concentration in the cytosol and lowers the effective cytosolic solute concentration. It follows that the driving force for solute diffusion from the pipette into the cell will increase as the cytosol is diluted. As solutes diffuse into the cell this leads to more water entering the cell through the membrane and more swelling.

Experimentally, the volume expansion usually reaches a steady-state, which allows cells to maintain a constant volume in the face of the osmotic gradient.

Imaging changes in cell volume.

Cells were visualized using a 63X objective with bright field or differential interference contract optics on a Zeiss Axiovert microscope and imaged using a CoolSnap HQ cooled CCD camera (Photometrics) and Metamorph software (Universal Imaging). Images were generally acquired 4/min. Cell volume was estimated by manually drawing a line around the perimeter of the cell in Metamorph, which calculated the cell area. Obviously, a two dimensional measurement of cell volume could introduce errors, especially in cells that had flat or irregular geometry. For these measurements, we chose cells that were relatively spherical and then lifted them slightly off the bottom of the dish. These suspended cells usually were reasonably symmetrical.

Cell surface biotinylation.

All reactions were carried out at 4 o C. Non-transfected HEK 293 cells and HEK 293 cells transfected with mBest2 were washed 3X with PBSB, placed in hypotonic or hypertonic medium for 15 min, then placed on ice and biotinylated with 0.5 mg/ml Sulfo-NHS-LC Biotin (Pierce, Rockford, IL, USA) in PBSB for 40 min. The cells were washed with PBSB, incubated in 100 mM glycine in PBSB to quench unreacted biotin, and washed 3X with PBSB, scraped from the dish and pelleted by centrifugation in a clinical centrifuge 3-5 min. The cell pellet was sonicated in Lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM HEPES pH 7.4, 1% Triton X-100, 0.5% protease inhibitor cocktail III (Calbiochem, San Diego, CA, USA), and 10 µM PMSF) and incubated 30 min. 250 µl of Lysis Buffer was used for each 100 mm dish of cells (<0.5 mg protein). The extract was clarified by centrifugation at 10,000 g for 15 min. 200 µl of extract was incubated with 50 µl of streptavidin beads (Pierce) overnight with gentle agitation. The beads were collected by centrifugation at 10,000 g for 10 min. The beads were washed 4X with 0.6 ml Lysis Buffer + 200 mM NaCl. The bound biotinylated proteins were eluted with 200 µl 2X Laemmli buffer. Protein samples were run on 4-15% gradient polyacrylamide gels in 25 mM Tris-HCl pH 8.3, 200mM glycine, 0.5% SDS with ~10 µg of protein per well.

The proteins were electrophoretically transferred to Hybond nitrocelluose membranes in 25 mM Tris-HCl pH 8.3, 200 mM Glycine, 20% methanol. The membranes were blocked with 5% dry milk in PBS with 0.1% Tween-20 (PBS-T) overnight at 4 o C or 1 hr at room temperature. Depending on the experiment, the nitrocellulose probed with a polyclonal antibody raised against a hBest1 C-terminal peptide AKQNVRGQEDNK in rabbits (1/1000 dilution) followed by horseradish peroxidase conjugated goat anti-rabbit IgG (1/4000) (Jackson Immuno Research, West Grove, PA, USA) in PBS-T with 1% dry milk. Immunoreactive or streptavidin-reactive bands were visualized by enhanced chemiluminescence (Pierce Biosciences). The reactive bands were quantified using an Alpha Innotech Fluorochem 8000 imaging system.

Solutions

Ringer solution used for mouse RPE cells contained (mM): 135 NaCl, 5 KCl, 10 glucose, 10 HEPES, 1 MgCl2, 1.8 CaCl2, pH 7.4 with NaOH. Isolation solution used for mouse RPE cells contained (mM): 135 NMDG, 5 KCl, 10 glucose, 3 EDTA, 3 Lcysteine, 10 HEPES, 1 MgCl2, 1.8 CaCl2, pH 7.4 with HCl. 'Low Ca' patch pipet solution contained (mM): 140 CsCl, 4 MgCl2, 0.062 CaCl2, 5 EGTA acid, 10 HEPES, 3.1 Na2-ATP, 0.42 Na2-GTP, pH 7.1 adjusted with CsOH. Calculated free Ca 2+ was 3.1 nM.

In the 'high Ca' pipet solution, CsCl was reduced to 133 mM and total CaCl2 was increased to 3.5 mM, so that the calculated free Ca 2+ was 624 nM. 'Normosmotic' (1T) extracellular solution contained (mM): 103.5 NaCl, 4 NaHCO3, 0.8 NaH2PO4, 5 pyruvic acid, 20 CsCl, 2 CaCl2, 1 MgCl2, 5 glucose, 10 HEPES, 30 mM mannitol, pH 7.4 with NaOH. Osmolality was 303 mOsm/Kg for both intra-and normosmotic extracellular solutions (Micro Osmometer, Model 3300; Advanced Instrument, Norword, MA).

'Hyposmotic' extracellular solution (0.9T, i.e. 274 mOsm/Kg) was obtained by omitting mannitol from the 1T solution. 'Hyperosmotic' extracellular solutions were obtained by increasing the mannitol concentration to 90 mM (1.2T) or 140 mM (1.36T). In some experiments, 'hyperosmotic' 1.2T solution was obtained by adding 50 mM NaCl to the 'hyposmotic' 0.9T solution.

Data Analysis

Currents were normalized to cell membrane capacitance, Cm. Mean values for Cm were: 10.4±0.4 pF (n=217) for HEK 293 cells; 36.2±4.2 pF (n=23) for HeLa cells; 26.7±4.6 pF (n=14) for ARPE-19 cells; 115.4±4.8 pF (n=69) for isolated mouse RPE cells. Although we did not measure the membrane capacitance systematically during the time course of an experiment, it appeared that cell capacitance did not change significantly during recording. On average Cm increased 10 ± 10% in hypertonic solution (n = 7) whereas hyposomotic solutions caused on average a 19 ± 7 % increase in Cm (n = 17). In any case, we do not believe that these small changes (<2 pF) in capacitance are reliable, considering that small changes in bath level or other stray capacitance could contribute to changes in measured Cm during the experiment. [START_REF] Ross | Membrane chloride conductance and capacitance in Jurkat T lymphocytes during osmotic swelling[END_REF] also report that during hypotonic swelling of Jurkat T-cells, changes in membrane capacitance are quite modest.

Data are expressed as mean ± s.e.m. Changes in current amplitude induced by cell shrinkage or swelling are expressed relative to control condition, each cell being its own control. Paired Student's t-test was used to test for statistical analysis, and a difference was considered statistically significant when p was <0.05.

RESULTS

Expression of hBest1 and mBest2 in various cell lines

HEK 293 cells transiently transfected with hBest1 or mBest2 were subjected to whole-cell patch-clamp using solutions that eliminated cation currents and set the Cl - equilibrium potential close to 0 mV. Free intracellular Ca 2+ (Cai) was buffered to either 3 nM (low Cai) or 620 nM (high Cai) with EGTA. Current densities in EGFP transfected HEK 293 cells at -100 mV were -2.0 7.0 pA/pF (n=4) in low Cai and -7.7  5.9 pA/pF (n=4) in high Cai. These values did not differ from non-transfected cells (n=5, not shown). Because the current values in WT and EGFP transfected cells were the same, these groups were usually combined (WT/GFP in Fig. 1).

In contrast, cells transfected with EGFP+hBest1 or EGFP+mBest2 exhibited a large Cl -current. As already reported in HEK 293 cells [START_REF] Sun | The vitelliform macular dystrophy protein defines a new family of chloride channels[END_REF][START_REF] Qu | Mouse bestrophin-2 is a bona fide Clchannel: Identification of a residue important in anion binding and conductance[END_REF], the current had a roughly linear I-V relationship, reversed at the expected Cl - equilibrium potential and was stimulated by Cai. Under these recording conditions, both hBest1 and mBest2 produced a Cl -current even with 3 nM free Cai (respectively, -46.4  19.6 pA/pF (n=16) and -94.3  26.6 pA/pF (n=13) at -100 mV) but the current amplitude doubled (with hBest1) or tripled (with mBest2) when Cai was increased to 620 nM (respectively, -105.7  17.5 pA/pF (n=26) and -364.5  92.9 pA/pF (n=14) at -100 mV).

At 620 nM, Cai was likely saturating bestrophin currents, as increasing Cai to 10 μM had no additional effect on hBest1 current amplitude (n=5, data not shown).

(Figure 1 near here)

As recently demonstrated for mBest2 [START_REF] Qu | Mouse bestrophin-2 is a bona fide Clchannel: Identification of a residue important in anion binding and conductance[END_REF], we found that transient expression of hBest1 also produced a Cl -current in two other human epithelial cell lines, ARPE-19 (Fig. 1C) and HeLa cells (Fig. 1D). These cells had a very small current when they were either not transfected or transfected with EGFP alone (-1.0  0.3 pA/pF (n=5) for ARPE-19 cells and -4.2 1.0 pA/pF (n=7) for HeLa cells, at -100 mV in high Cai), but the current strongly increased when transfected with hBest1 (-23.9  8.3 pA/pF (n=9) for ARPE-19 cells and -13.9  3.5 pA/pF (n=9) for HeLa cells). Although the Cai dependence of the hBest1-induced current was not examined in ARPE-19 and HeLa cells, the I-V relationship obtained in high Cai was very similar to that obtained in HEK 293 cells. The similarity in the hBest1 induced current in three different cell lines strengthens the likelihood that the current was encoded by hBest1, and not the result of an up-regulation of an endogenous current. Also, transfection of HEK 293 and HeLa cells with the G299E mutant of hBest1, a point mutant associated with Best disease, did not induce currents: current amplitudes were not different from EGFP/WT controls (-1.9  1.0 pA/pF (n=4) for HEK 293 cells and -3.7  1.7 pA/pF (n=7) for HeLa cells, both at -100 mV and in high Cai).

We have previously shown, using immunocytochemistry and cell surface biotinylation that mBest2 and xBest2 are expressed on the cell surface in transiently transfected cells [START_REF] Qu | Two bestrophins cloned from Xenopus laevis Oocytes express Ca-activated Cl currents[END_REF][START_REF] Qu | Mouse bestrophin-2 is a bona fide Clchannel: Identification of a residue important in anion binding and conductance[END_REF]. To determine whether hBest1 is expressed on the cell surface, we biotinylated intact cells with a membrane-impermeant biotinylation reagent (Fig. 2). Approximately 12% of the total hBest1 was found on the cell surface under these conditions. This agrees with our immunocytochemical data (unpublished) and data of [START_REF] Tsunenari | Structure-function analysis of the bestrophin family of anion channels[END_REF] that the majority of hBest1 is located in intracellular membranes. However, the fraction of hBest1 at the cell surface does not appear to be strongly affected by the osmotic conditions (Fig. 2).

(Figure 2 near here)

Effect of hyperosmotic cell shrinkage on hBest1 currents in HEK 293 cells

Figure 3 shows an experiment performed on a HEK 293 cell transfected with EGFP+hBest1 with high Cai in the patch pipet. Under normosmotic conditions (1T), a large, time independent current was observed (Fig. 3A, top traces). When 60 mM mannitol was added to the extracellular solution, which increased the osmolality by 20% from 303 to 364 mOsm/Kg (1.2T), the current was strongly inhibited (Fig. 3A, middle traces). This was accompanied by a clear shrinkage of the cell as indicated by the micrograph next to the current traces. The current inhibition was reversible upon a subsequent reduction in the osmolality from 364 to 274 mOsm/Kg (0.9T), and this was accompanied by an increase in cell volume (Fig. 3A, bottom traces). The time course of the current response to hyperosmotic shrinkage and hyposmotic swelling was followed using a repetitive ramp protocol as described in Fig. 3B. Current amplitudes at -100 and +100 mV were continuously measured every 8 sec at the beginning and end of each 1.35sec duration voltage ramp (Fig. 3C). Hyperosmotic shrinkage induced a progressive inhibition of hBest1 current at both potentials, with 50% inhibition occurring within ~4 min. In this cell, hyperosmotic shrinkage reduced the current by 90% within 10 min (compare trace b and a in Fig. 3B). The current recovered its amplitude following a similar slow time course when the cell was exposed to 0.9T solution. A steady-state was obtained at ~15 min in hyposmotic solution, and hBest1 current was ~10% above its initial amplitude. Finally, the cell was returned to normosmotic 1T solution, and the current returned to its initial amplitude.

(Figure 3 near here)

The effect of hyperosmotic shrinkage in high Cai was tested in a total of 10 HEK 293 cells expressing hBest1+EGFP obtained from 3 different cultures. In 4 of these cells, the hyperosmotic 1.2T solution was made by increasing the NaCl concentration by 30 mM. In the other 6 cells, osmolality was increased by adding 60 mM mannitol. The results were the same in both conditions. On average, hBest1 current decreased by 71.2  6.0 % at -100 mV and 70.2  5.4 % at +100 mV when exposed for 6.1  1.0 min to a 1.2T solution (p<0.002, n=10, Fig. 9). Fig. 4 shows the average current traces (normalized to cell membrane capacitance) obtained in normosmotic 1T and hyperosmotic 1.2T solution during the standard ramp protocol (Fig. 4A) and the average current difference for the two conditions (Fig. 4B). Hyperosmotic solutions reduced the amplitude of the current equally at each membrane potential, so that the I-V relationships for the 1T solution, the 1.2T solution and the difference current had similar shapes. The normalized I-V curves were nearly superimposable (not shown).

(Figure 4 near here)

Effect of hyposmotic cell swelling on hBest1 currents in HEK 293 cells

The next series of experiments were aimed at testing whether hyposmotic solution alone had any effect on hBest1 current. As HEK 293 cells possess an endogenous swelling-activated Cl -current (ICl(swell)) or volume-regulated anion channel (VRAC) [START_REF] Nilius | Amazing chloride channels: an overview[END_REF][START_REF] Sardini | Cell volume regulation and swelling-activated chloride channels[END_REF], we first examined the effect of 0.9T hyposmotic swelling on WT/GFP cells. When WT/GFP HEK 293 cells were exposed to hyposmotic 0.9T solution, a swelling activated VRAC current was observed in 8 out of 16 cells, and its amplitude was small, averaging 6.23.1 pA/pF at -100 mV and 5.13.2 pA/pF at + 100 mV. These values are smaller than those reported by other investigators who have used more severe hyposmotic conditions. Increases of ~20 pF/pA have been reported for 0.6 T -0.7 T solutions. A similar amplitude of VRAC current was found in HEK 293 cells transfected with the dominant negative G299E mutant of hBest1 (n=4, not shown). As the amplitude of VRAC current was small with respect to the amplitude of hBest1 current, we anticipated that VRAC might cause little interference with the effect of hyposmotic swelling on hBest1 current. We then exposed HEK 293 cells expressing hBest1+EGFP to a hyposmotic 0.9T solution while continuously recording the current elicited by voltage ramps. The response varied greatly from cell to cell: 6 out of 8 cells in low Cai and 16 out of 35 cells in high Cai responded by an increase >10% in total current, Interestingly, in the hBest1 cells that did not respond, the current density was on average >2-fold larger (p<0.05) than the cells that did respond. Fig. 5 summarizes the results of the 16 cells responding to hyposmotic solution with high Cai. When these cells were exposed to 0.9T solution, the current progressively increased to reach a steady-state at 6.1  0.9 min (n=16). Shown in Fig. 5 are the average current traces (normalized to cell membrane capacitance) obtained in normosmotic 1T and hyposmotic 0.9T solution during the standard ramp protocol (Fig. 5A) and the average current difference for the two conditions (Fig. 5B). While the average ramp I-V relationships had similar linear shapes in normosmotic and hyposmotic conditions, the currents during the 500 ms steps before and after the ramp clearly differed. Unlike the hBest1 current in 1T condition which was time-independent, the currents in 0.9T showed a time-dependent inactivation at +100 mV, and recovery from inactivation at -100 mV.

The individual current traces obtained during a step protocol (inset in Fig. 5A) further illustrate the time-dependent behavior of the currents in hyposmotic solution. This current response of hBest1 transfected HEK 293 cells to hyposmotic swelling had all the characteristic features of a VRAC current [START_REF] Sardini | Cell volume regulation and swelling-activated chloride channels[END_REF]. However, its amplitude was 8-10 fold larger than the VRAC current in WT/GFP cells, suggesting that moderate expression of hBest1 may facilitate the development of, or contribute to, a VRAC current.

(Figure 5 near here)

In an attempt to separate hBest1 and VRAC currents, current amplitudes were monitored at the beginning and end of the 500-msec steps to -100 mV and +100 mV, which preceded and followed each ramp protocol. A difference between the amplitudes at the onset and at the end of the pulse indicates a time dependent component in the total current. Because the hBest1 current is time-independent, this measurement allows us to separate, at least qualitatively, hBest1 and VRAC currents. Figure 6 shows a typical experiment performed on an HEK 293 cell transfected with EGFP+hBest1 with low Cai in the patch pipet. At the beginning of the experiment, when the cell was in normosmotic solution (trace a), a typical hBest1 current was observed, with no time dependence at either -100 or +100 mV. Exposing the cell to a hyposmotic 0.9T solution induced a biphasic increase in the current amplitude. During the first ~2 minutes, the current increased to ~60% above control, but remained essentially time-independent (trace b), suggesting that this increase was due to a rise in hBest1 current. The current remained essentially stable at this level for an additional ~2 min and then resumed increasing.

During this second phase of increase, the current developed time-dependent activation and inactivation (trace c). This time-dependence indicates that activation of VRAC was responsible for this second and more robust increase in current. After hyposmotic swelling, the cell was exposed to a 1.2T hyperosmotic solution and the stimulatory effect disappeared as well as the time dependence (trace d). After ~6 min in 1.2T solution, only a time-independent current remained, whose amplitudes at -100 and +100 mV were only ~40% those in 1T, indicating inhibition of hBest1 current.

The effects of hyposmotic swelling on hBest1 (time-independent) and VRAC (time-dependent) currents were clearly separated in a total of 7 cells (5 in low Cai and 2 in high Cai). In these cells, hyposmotic swelling led to a stimulation of hBest1 current which varied from 30 to 70%. However, due to the uncertainty in the relative contribution of each current and the poor pharmacology of Cl -channels, no attempt was made to further quantify these effects.

(Figure 6 near here) Relationship of hBest1 current amplitude to cell volume.

Figure 3 shows that hyperosmolar solutions produce both cell shrinkage and a decrease in hBest1 current. To determine whether the change in cell volume and hBest1 current occurred with the same time course, we imaged cells at 15 second intervals during a voltage clamp experiment. In the experiment in Figure 7, cell volume and hBest1 changed with virtually identical time courses. In the three cells we examined in detail, the current decreased with a τ = 1.6 ± 0.2 min and the cell volume changed with a τ = 1.5 ± 0.2 min.

(Figure 7 near here)

Effect of osmolality on mBest2 currents in HEK 293 cells

The above results indicate that the hBest1 current is sensitive to osmolality and its expression promotes VRAC activation by cell swelling. To examine whether these effects were specific to hBest1, we repeated the same type of experiments in HEK 293 cells transfected with mBest2. Figure 8 shows a typical experiment performed in high Cai.

Under normosmotic condition (1T), a large, essentially time-independent mBest2 current was observed (Fig. 8A, trace a). Exposure of the cell to 1.2T hyperosmotic solution strongly inhibited this current by ~80% within 8 min (trace b). Upon application of a hyposmotic solution, the current slowly increased again, and about 40% of the inhibition recovered within ~10 min (trace c). Interestingly, hyposmotic swelling did not elicit any time-dependent VRAC current, as the current amplitudes at the beginning and end of the 500-msec steps to -100 mV and +100 mV were very similar throughout the entire 30 min experiment (Fig. 8B). In a total of 20 mBest2 expressing HEK 293 cells tested (11 in low Cai and 9 in high Cai), hyposmotic swelling, tested either alone (12 cells) or after hyperosmotic shrinkage (8 cells) never induced a time-dependent current. Hyposmotic swelling had no effect on the time-independent (mBest2) current in high Cai (n=6, Fig. 9) but increased the current in 3 out of 6 cells in low Cai by 40 to 60%. After hyperosmotic shrinkage, hyposmotic swelling increased mBest2 current back to ~70% of its initial amplitude in normosmotic condition in low Cai (n=5, Fig. 9), but had no effect in high Cai (n=3, Fig. 9). These results indicate that, like hBest1, mBest2 is sensitive to cell volume. However, unlike hBest1, expression of mBest2 did not promote VRAC activation by cell swelling.

(Figure 8 near here)

The fact that Cl -currents in HEK 293 cells expressing hBest1 or mBest2 are sensitive to cell volume changes suggests that bestrophin currents are volume-sensitive.

However, another possibility is that bestrophin expression up-regulates an endogenous current or alters the trafficking of an endogenous channel to the plasma membrane, which would show a volume-sensitivity. To test this hypothesis, hBest1 was expressed in two other cell lines. We assumed that if the volume-sensitivity of the hBest1 current was the same in different cell lines, this would strengthen the likelihood that it is the current encoded by hBest1 and not an endogenous current which is volume-sensitive. As shown above (Fig. 1), expression of hBest1 in HeLa cells produced a hBest1 current that was similar to the one in HEK 293 cells. Currents in HeLa cells were also sensitive to hyperosmotic shrinkage (not shown).

Figure 9 summarizes our results. Hyperosmotic shrinkage consistently inhibited by 50-70% hBest1 current in HEK 293, ARPE-19 and HeLa cells, and mBest2 current in HEK 293. In HEK 293 cells where most experiments were performed, we examined whether intracellular Ca played a role, but found no significant difference in the degree of inhibition by hyperosmotic shrinkage of mBest2 and hBest1 currents at 3 nM, 620 nM or 10 μM Cai. As shown above, the effect of hyposmotic swelling on hBest1 current was difficult to quantify because of the interference of VRAC current. In contrast, in mBest2 expressing cells, where VRAC was not activated, hyposmotic swelling increased mBest2 current in 50% of the cells in low Cai, although on average the effect was not significant.

When hyposmotic swelling followed hyperosmotic shrinkage, mBest2 current recovered most of its initial amplitude in low Cai but not in high Cai.

(Figure 9 near here)

Effect of hyperosmotic shrinkage on CFTR current

To evaluate the degree of specificity of the effects of volume changes on bestrophin currents, we examined the effect of hyperosmotic shrinkage on another Cl - current unrelated to bestrophin family. For this, HEK 293 cells were transfected with the human cystic fibrosis transmembrane conductance regulator (hCFTR) cDNA and wholecell patch-clamped in high Cai. The hCFTR current was activated by 50 μM forskolin, a direct activator of adenylyl cyclase, and 1 mM 3-isobutyl-1-methylxanthine (IBMX), a broad spectrum phosphodiesterase inhibitor. Under these conditions, Cl -current density was -428.8  140.5 pA/pF at -100 and 496.4  164.9 pA/pF +100 mV (n=10). Although the current density was comparable to that observed in mBest2 transfected cells (see Fig. 1), hyperosmotic shrinkage (1.2T) had no effect on hCFTR current. Indeed, application of a 1.2T solution during 7.0  2.1 min resulted in a -9.1  3.9 % and -1.9  5.4 % change in hCFTR current amplitude at -100 and +100 mV, respectively (n=6). These results indicate that the volume sensitivity of the bestrophin currents is not simply the consequence of the high transmembrane Cl -fluxes that these channels carry, but is rather due to some specific effect on the channel itself.

Effect of cell volume changes on Cl -current in mouse RPE cells

As no animal model of Best disease is available yet, we cannot examine how a mutation in bestrophin 1 affects the function of native RPE cells. However, the above results indicate that bestrophin currents are highly sensitive to hyperosmotic shrinkage. This led us to investigate whether Cl -currents in native RPE cells also possess such feature. Freshly isolated mouse RPE cells were subjected to whole-cell patch-clamp using identical solutions and voltage protocols as used in the experiments on HEK 293 cells. In the experiment shown in Fig. 9, a mouse RPE cell was first exposed to normosmotic solution. The current traces obtained during a step voltage-clamp protocol (Fig. 10A) show essentially time-independent currents, except at very negative potentials where a time-dependent activation was observed. The reversal potential of the current was near 0 mV, which is near the estimated equilibrium potential for Cl -ions. Application of a hyposmotic 0.9T solution strongly increased the current amplitude at every potential. The current outwardly rectified and exhibited a clear inactivation time course at +100 mV. This current increase was strongly inhibited by addition of niflumic acid (NFA, 100 μM) and anthracene-9-carboxylic acid (9-AC, 500 μM), two classical blockers of the VRAC channel. The time course of the current response to hyposmotic swelling was followed using the repetitive ramp protocol as described in Fig. 10B. Current amplitudes at -100 and +100 mV were measured every 8 sec at the beginning and end of each 1.35-sec duration voltage ramp (Fig. 10C). Hyposmotic swelling increased mouse RPE Cl - currents over a period of 8 min, and addition of 9-AC+NFA reduced this stimulation by ~80% within 6 min. These results demonstrate the presence of a swelling-activated VRAC current in mouse RPE cells.

(Figure 10 near here)

The effect of hyperosmotic shrinkage was subsequently examined. Figure 11 shows an experiment in which a mouse RPE cell was successively exposed to normosmotic (1T), hyperosmotic (1.36T) and hyposmotic (0.9T) solutions while voltageclamped using the ramp protocol. The micrographs in Fig. 11A indicate that the cell underwent the expected volume decrease and increase during the hyperosmotic and hyposmotic stimuli, respectively. During hyperosmotic shrinkage, there was a progressive inhibition of the current until a new steady-state was reached within ~6 min (Fig. 11B &C). The Cl -current decreased by ~60% at +100 mV and decreased by ~40% at -100 mV. When the extracellular solution was switched to a hyposmotic solution (0.9T), the current slowly increased at +100 mV to return to its control level within 20 min. However, the effect at -100 mV was smaller, and the current amplitude returned to only ~50% of its control amplitude within the same period.

(Figure 11 near here)

The results of several similar experiments are summarized in Fig. 12. The I-V curves in Fig. 12A demonstrate that total current density was reduced in 1.36T hyperosmotic shrinkage and increased in 0.9T hyposmotic swelling. Both maneuvers affected the current at each membrane potential, but the effects were more pronounced at positive potentials. Data from paired experiments (Fig. 12B), using each cell as its own control, show that a moderate (1.2T) hyperosmotic shrinkage reduced the current by only 20% both at negative and positive potentials. However, a stronger hyperosmotic shrinkage (1.36T) reduced the current by 50% at +100 mV, but only by ~30% at -100 mV. In both situations, exposing the cell to a hyposmotic 0.9T solution increased the current above control level, and the effect was larger at +100 than -100 mV. To make sure that current in normosmotic solution was not contaminated by VRAC current, we tested the effect of 9-AC (500 μM) + NFA (100 μM) under control conditions. In 7 mouse RPE cells, current amplitude decreased by -13.88.7% at -100 mV and -18.930.2% at +100 mV, and the effects were not statistically significant (p>0.2).

(Figure 12 near here)

DISCUSSION

In this paper, we show that expression of either hBest1 and mBest2 in several cell lines induces a novel Cl -current that is strongly inhibited by hyperosmotic solutions and stimulated, but non-systematically and to a lesser extent, by hyposmotic solutions. The latter effect could not be quantified because of a simultaneous activation of a VRAC current.

Stretch-activated or volume-regulated ion channels are thought to play a key role in regulating cell volume [START_REF] Nilius | Properties of volume-regulated anion channels in mammalian cells[END_REF][START_REF] Hoffmann | Membrane mechanisms in volume and pH regulation in vertebrate cells[END_REF][START_REF] Lang | Functional significance of cell volume regulatory mechanisms[END_REF]. The canonical volume-regulated anion channel (VRAC) is outwardly-rectifying and exhibits time-dependent inactivation at positive potentials and is stimulated by hyposmotic solutions. In non-transfected HEK 293 cells, we find that the endogenous VRAC current is small (~6 pA/pF) in 0.9T hyposmotic solutions. Other investigators who have studied this current in HEK cells (e.g., [START_REF] Nilius | Differential activation of the volume-sensitive cation channel TRP12 (OTRPC4) and volumeregulated anion currents in HEK-293 cells[END_REF][START_REF] Sardini | Cell volume regulation and swelling-activated chloride channels[END_REF]) have observed larger VRAC currents, but have used considerably lower osmolality solutions (0.7T -0.8T). For our discussion, we define VRAC as an outwardly-rectifying, time-dependent current.

Are bestrophins candidates for VRACs?

The VRAC current was about 10-fold larger in hBest1-transfected cells than in non-transfected cells. On this basis, one might be tempted to make the snap conclusion that hBest1 is a component of VRAC. However, the muddled history of the identification of the molecular counterpart of VRAC makes us loathe to even consider this conclusion [START_REF] Sardini | Cell volume regulation and swelling-activated chloride channels[END_REF][START_REF] Nilius | Properties of volume-regulated anion channels in mammalian cells[END_REF]. p-Glycoprotein, ClC-3, ClC-2, pICln, phospholemman, and the Cl-HCO3 exchanger have all been proposed as VRACs, but none of these has received acceptance. It seems likely that these proteins somehow regulate the expression or function of endogenous VRAC channels. We think that hBest1 probably also somehow increases endogenous VRAC expression or function. The observation that mBest2 expression does not increase VRAC current supports this suggestion.

There are a number of reasons to think that bestrophins are not VRAC channels, and are rather a different kind of Cl -channel that is both Cai and volume sensitive and may therefore play a different role in cell volume regulation. Reasons for thinking that bestrophins are not canonical VRACs include the following: The kinetics and rectification of VRAC are distinctly different than the bestrophin currents in normosmotic solution. Bestrophin currents are time-independent and have linear IV relationships. Furthermore, the bestrophin current, especially the mBest2 current, is rather insensitive to hyposmolality. Rather, the bestrophin currents are strongly inhibited by hyperosmotic solutions. Quantification of the effects of hyposmolality on bestrophin currents was complicated because of the difficulty in separating it from the endogenous VRAC, but if one accepts the difference in kinetics and rectification as sufficient evidence for differentiating between the two currents, it seems that the bestrophin current is less sensitive to hyposmolality than the endogenous VRAC.

The rather small and non systematic effect of hyposmolality on bestrophin currents does not necessarily exclude their being regulated by cell swelling. First, we used relatively modest hyposmotic conditions. Bestrophin currents might be stimulated by more extreme conditions. Second, the ability of cells to respond to hyposmolality depends somehow on the "set-point" of the cell. For example, the set-point can be altered by the ionic strength of the cytosol: higher ionic strengths inhibit the ability of VRAC to be stimulated by swelling [START_REF] Cannon | Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel[END_REF][START_REF] Nilius | Activation of volume-regulated chloride currents by reduction of intracellular ionic strength in bovine endothelial cells[END_REF]. Further, keeping cells for prolonged periods in different ionic conditions alters the sensitivity to RVD and RVI [START_REF] Lang | Functional significance of cell volume regulatory mechanisms[END_REF]. However, it seems that bestrophin currents are less sensitive to swelling than VRAC. As shown in Fig. 5, upon shifting to 0.9T, the change in the timedependent current (VRAC) is slower but larger than the change in the time-independent current (hBest1).

An additional argument against the contribution of bestrophins to VRAC is the fact that the activation of canonical VRAC is usually considered to be independent of Ca [START_REF] Nilius | Properties of volume-regulated anion channels in mammalian cells[END_REF]. However, it has been reported that in some cells a permissive low level of Ca (50 nM) may be required for VRAC activation [START_REF] Nilius | Properties of volume-regulated anion channels in mammalian cells[END_REF][START_REF] Szucs | Activation of the volumesensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration[END_REF]. Increases in cell volume are often accompanied by increases in Cai, but the role of Cai remains controversial and unresolved [START_REF] Mccarty | Calcium signaling in cell volume regulation[END_REF]. The consensus is that increases in Cai are not obligatory for regulatory volume decrease, but may facilitate it [START_REF] Altamirano | Regulatory Volume Decrease and Intracellular Ca 2+ in Murine Neuroblastoma Cells Studied with Fluorescent Probes[END_REF].

Cell volume regulation in mouse RPE cells. Mouse RPE cells clearly have

volume regulated Cl -currents. A component of the Cl -current in these cells is inhibited by cell shrinkage. However, because of the absence of suitable pharmacological tools, it is not possible to determine whether this current is due to bestrophin. The current differs in some respects from the mBest2 or hBest1 in that it outwardly rectifies. Unfortunately, we have not yet studied mBest1, which may have other properties. In any case, the volume sensitive currents are likely to be important in the function of RPE cells in the context of the retina. For example, light produces changes in the volume and ionic composition of the sub-retinal space [START_REF] Huang | Light-evoked expansion of subretinal space volume in the retina of the frog[END_REF]) that might result in osmotic stresses on the RPE.

In addition to regulating the composition of the subretinal space, the RPE also is involved in phagocytosis of shed photoreceptor discs [START_REF] Besharse | The daily light-dark cycle and rhythmic metabolism in the photoreceptor pigment epithelial complex[END_REF][START_REF] Lavail | Outer segment disc shedding and phagocytosis in the outer retina[END_REF][START_REF] Nguyen-Legros | Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium[END_REF]. The process of ingestion of photoreceptor discs by RPE cells involves substantial changes in RPE cell morphology and volume, both of which may be dependent on Cl -channels. Engulfment of the photoreceptor discs may involve large changes in the volume of the RPE cell. It has been estimated that each RPE cell ingests >25,000 discs per day [START_REF] Nguyen-Legros | Renewal of photoreceptor outer segments and their phagocytosis by the retinal pigment epithelium[END_REF]. In liver cells, phagocytosis is accompanied by large osmolyte fluxes via anion channels and transporters [START_REF] Wettstein | Release of osmolytes induced by phagocytosis and hormones in rat liver[END_REF]. (1.36T) or hyposmotic (0.9T) solutions. B, Cl -current density was recorded at -100 mV (bottom histograms) or +100 mV (top histograms). In all experiments, the cells were first exposed to normosmotic solution (1T), then to hyperosmotic (1.2T or 1.36T) solutions, and finally some cells were exposed to hyposmotic solution (0.9T). The changes in current densities were normalized to that obtained in 1T (-100% at -100 mV; +100% at +100 mV). All experiments were performed with 620 nM Cai.

  Fig. 1A & B show average currentvoltage (I-V) relationships obtained with slow ramp protocols (see Methods) in HEK 293 cells transfected with either EGFP alone, EGFP+hBest1 or EGFP+mBest2. In HEK 293 cells transfected with EGFP alone, currents were consistently small regardless of Cai.

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. hBest1 is expressed on the cell surface.Transiently transfected HEK 293 cells were biotinylated as described in Methods. Inset: Western blot. Cells were bathed in hypotonic medium (lanes 1 and 2) or hypertonic medium (lanes 3 and 4). Lanes 1 and 3 are biotinylated hBest1. Lanes 2 and 4 are nonbiotinylated. The fraction of hBest1 biotinylated by cell surface biotinylation reagent is quantified in the graph (mean of 3 experiments).

Figure 3 .

 3 Figure 3. Effect of hyperosmotic cell shrinkage on hBest1 currents in HEK 293 cells Patch pipet contained high Cai solution. A, individual current traces obtained in response to the step protocol indicated on top. Top traces: normosmotic, 1T. Middle traces: hyperosmotic, 1.2T. Bottom traces: hyposmotic, 0.9T. Corresponding changes in cell volume are illustrated by the micrographs. B, Ramp protocol and corresponding current traces. C, Time course of the current amplitudes at -100 mV (circles) and +100 mV

Figure 4 .

 4 Figure 4. Effect of hyperosmotic cell shrinkage on hBest1 currents in HEK 293 cells A, Average current traces (thick lines, normalized to cell membrane capacitance) and s.e.m. (thin lines) of 12 HEK 293 cells transfected with EGFP+hBest1 and exposed first to normosmotic 1T and subsequently to hyperosmotic 1.2T solutions during the standard ramp protocol. The cells were whole-cell voltage-clamped with high Cai in the patch pipet. The x-axis indicates the changes in membrane potential during the ramp protocol. B, Average current difference (thick line) and s.e.m. (thin line) for the two conditions shown in A.

Figure 5 .

 5 Figure 5. Effect of hyposmotic cell swelling on hBest1 currents in HEK 293 cells A, Average current traces (thick lines, normalized to cell membrane capacitance) and s.e.m. (thin lines) of 16 HEK 293 cells transfected with EGFP+hBest1 and exposed first to normosmotic 1T and subsequently to hyposmotic 0.9T solutions during the standard ramp protocol. The cells were whole-cell voltage-clamped with high Cai in the patch pipet. The x-axis indicates the changes in membrane potential during the ramp protocol.The inset shows individual current traces obtained for one of these cells in response to the

Figure 6 .

 6 Figure 6. Effect of hyposmotic cell swelling on hBest1 currents in HEK 293 cells Patch pipet contained low Cai. A, Ramp protocol and current traces at times indicated by the corresponding letters in B. B, Time course of the current amplitudes at -100 mV (circles) and +100 mV (triangles) to hyperosmotic shrinkage and hyposmotic swelling at times indicated by the corresponding symbols in A. The cell was first exposed to normosmotic (1T) solution, and then to hyposmotic (0.9T) and hyperosmotic (1.2T) solutions as indicated by the solid lines. Cell membrane capacitance was 22.0 pF.

Figure 7 .

 7 Figure 7. Time course of change in cell volume and hBest1 current. Patch pipet contained high Cai. The cell was exposed to hyperosmotic (1.2T) solution at the arrow. Cell area was measured from high-resolution images and current amplitudes from voltage clamp ramps.

Figure 8 .

 8 Figure 8. Effect of osmolality on mBest2 currents in HEK 293 cells Patch pipet contained high Cai. A, Ramp protocol used and individual current traces obtained at times indicated by the corresponding letters in B. B, Time course of current amplitudes at -100 mV (circles) and +100 mV (triangles) in response to hyperosmotic shrinkage and hyposmotic swelling at times indicated by the corresponding symbols in A.The cell was first exposed to normosmotic (1T) solution, and then to hyperosmotic (1.2T) and hyposmotic (0.9T) solutions as indicated by the solid lines. Cell membrane capacitance was 17.6 pF.

Figure 9 .

 9 Figure 9. Summary of the effects of cell volume changes on hBest1 and mBest2 currents Cells were exposed to normosmotic solution (1T) and bestrophin current density was recorded at -100 mV (bottom histograms) or +100 mV (top histograms). The cells were subsequently exposed to either hyperosmotic (1.2T) or hyposmotic (0.9T) solutions, and the changes in current densities were normalized to that obtained in 1T (-100% at -100 mV; +100% at +100 mV). When indicated by 0.9T  1.2T, the cells experienced a hyposmotic challenge after the initial hyperosmotic one. Summarized are experiments performed in: HEK 293 cells transfected with hBest1 + EGFP and whole-cell voltageclamped with 3 nM, 620 nM or 10 μM Cai; HEK 293 cells transfected with mBest2 + EGFP and with 3 nM or 620 nM Cai; HeLa and ARPE-19 cells transfected with hBest1 + EGFP and with 620 nM Cai.

Figure 10 .

 10 Figure 10. Effect of hyposmotic cell swelling on Cl -current in mouse RPE cells Patch pipet contained high Cai. A, current traces obtained in response to the step protocol indicated on top. Top traces: normosmotic, 1T. Middle traces: hyposmotic, 0.9T. Bottom traces: hyposomotic (0.9T) in presence of niflumic acid (NFA, 100 μM) and anthracene-9-carboxylic acid (9-AC, 500 μM). B, Ramp protocol and current traces. C, Time course of the current response to hyposmotic swelling and inhibition by NFA+9-AC. The cell was first exposed to normosmotic (1T), and subsequently to hyposmotic (0.9T) without or with NFA (100 μM) and 9-AC (500 μM) as indicated by the solid lines in C. The current traces in B were obtained at times indicated by the corresponding letters in C. Cell membrane capacitance was 87.2 pF.

Figure 11 .

 11 Figure 11. Effect of cell volume changes on Cl -current in mouse RPE cells Patch pipet contained high Cai. A, Micrographs of the cell exposed to normosmotic (1T), hyperosmotic (1.36T) and hyposmotic (0.9T) solutions. B, Ramp protocol and current traces. C, Time course of the current response to hyperosmotic shrinkage and hyposmotic swelling. The cell was exposed to normosmotic (1T), hyperosmotic (1.36T), and hyposmotic (0.9T) solutions as indicated by the solid lines in C. The micrographs in A and the individual current traces in B were obtained at times indicated by the corresponding letters in C. Cell membrane capacitance was 110.9 pF.

Figure 12 .

 12 Figure 12. Summary of the effects of cell volume changes on Cl -currents in mouse RPE cells A, Current-voltage (I-V) curves for the Cl -currents in isolated mouse RPE cells. Cells were first exposed to normosmotic solution (1T) and subsequently to either hyperosmotic

ACKNOWLEDGEMENTS

The authors thank Chun Pfahnl and Toni Grim for excellent technical assistance.

Supported by NIH grants GM60448 and EY014852. R. Fischmeister also received support from INSERM.