Published online before print September 5, 2008, doi: 10.1161/CIRCRESAHA.108.178517
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© 2008 American Heart Association, Inc.
Cellular Biology |
Bestrophin-3 (Vitelliform Macular Dystrophy 2–Like 3 Protein) Is Essential for the cGMP-Dependent Calcium-Activated Chloride Conductance in Vascular Smooth Muscle Cells
From the Water and Salt Centre (V.V.M., E.V.B., D.M.B.B., V.G., C.A., H.N.), Institute of Physiology and Biophysics; Institute of Anatomy (A.R.); and Department of Molecular Biology and Interdisciplinary Nanoscience Center (P.L., F.S.P.), University of Aarhus, Denmark. Present address for V.G. and H.N.: Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Sweden.
Correspondence to V. V. Matchkov, Institute of Physiology and Biophysics, University of Aarhus, 8000 Aarhus C, Denmark. E-mail vvm{at}fi.au.dk
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Abstract |
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Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca2+-activated Cl– currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca2+-activated Cl– current with unique characteristics in smooth muscle cells. This novel current has been shown to coexist with a "classic" (cGMP-independent) Ca2+-activated Cl– current and to have characteristics distinct from those previously known for Ca2+-activated Cl– currents. Here, we suggest that a bestrophin, a product of the Best gene family, is responsible for the cGMP-dependent Ca2+-activated Cl– current based on similarities between the membrane currents produced by heterologous expressions of bestrophins and the cGMP-dependent Ca2+-activated Cl– current. This is supported by similarities in the distribution pattern of the cGMP-dependent Ca2+-activated Cl– current and bestrophin-3 (the product of Best-3 gene) expression in different smooth muscle. Furthermore, downregulation of Best-3 gene expression with small interfering RNA both in cultured cells and in vascular smooth muscle cells in vivo was associated with a significant reduction of the cGMP-dependent Ca2+-activated Cl– current, whereas the magnitude of the classic Ca2+-activated Cl– current was not affected. The majority of previous suggestions that bestrophins are a new Cl– channel family were based on heterologous expression in cell culture studies. Our present results demonstrate that at least 1 family member, bestrophin-3, is essential for a well-defined endogenous Ca2+-activated Cl– current in smooth muscles in the intact vascular wall.
Key Words: bestrophins • calcium-activated chloride channel • cGMP • posttranscriptional regulation • vascular biology
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Introduction |
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Chloride channels are of key importance for several cellular functions, including control of membrane potential, volume regulation, and transepithelial water and salt transport. We1 and Large and colleagues2 recently described a novel Ca2+-activated Cl– current in smooth muscle with a dependence on cGMP and with characteristics distinct from the other known Ca2+-activated Cl– channels. It is of interest that cGMP, which causes vascular smooth muscle relaxation, can also activate a Cl– channel, which results in depolarization in vascular smooth muscle. Furthermore, the current is of similar magnitude as "classic" Ca2+-activated Cl– currents in most vascular beds and even larger in some vascular smooth muscles.3 It is, therefore, highly desirable to know the molecular structure of the channel responsible for this current because it is likely to play an important role in smooth muscle function.
Although their biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) are well established,4–6 the molecular identity of Ca2+-sensitive Cl– channels is still controversial.7 Recently, the gene responsible for vitelliform macular dystrophy8 and its homologs that code for bestrophin proteins have been suggested as candidates.9,10 Four bestrophin family members in the mammalian genome and many homologues in genomes of invertebrates and even prokaryotes have been identified.11–13 Two different nomenclatures for mammalian bestrophins were previously developed12,14: VMD2, VMD2-like1, VMD2-like3, and VMD2-like2 correspond to Best-1, -2, -3, and -4 (Table I in the online data supplement, available at http://circres.ahajournals.org). To avoid confusion, the HUGO and the Mouse Genome Database nomenclature committees have recently recommended using the Best nomenclature.
The majority of suggestions that bestrophins function as Cl– channels are based on the findings that expression of the gene in different cell types leads to the appearance of a Cl– conductance9,10 and that mutations or chemical modifications of the predicted channel pore change this conductance.15–18 Although downregulation by small interfering (si)RNA in recent studies demonstrated a direct association between the endogenous Cl– current in epithelial cell culture and Best-1 expression,19–21 the exact role of the bestrophins in native tissues remains questionable.13 An alternative suggestion is that bestrophins function as Ca2+ channel modulators based on the observation that heterologous expressed Best-1 modulates the kinetics of L-type Ca2+ channels.22 A recent study demonstrated that the ability to regulate L-type Ca2+ channels in addition to functioning as Cl– channels is a unique property of the Best-1 isoform.23
The recently described cGMP-dependent Ca2+-activated Cl– conductance shares many characteristics with the Cl– conductance seen after heterologous expression of Best genes, such as high [Ca2+]i sensitivity, linear voltage dependence, and anion permeability.9,10,13,15–17 Furthermore, dependence on cGMP and inhibition by micromolar Zn2+ concentrations that characterize the novel Cl– current is compatible with the molecular structure of the bestrophins.9,10,20,24–27 On this background, we set out to test the hypothesis that the novel cGMP-dependent Ca2+-activated Cl– current is mediated by one of the Best gene family products.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Small Interfering RNAs
Two siRNAs directed against exons 7 and 10 (of rat Best-3 gene [Gene ID 3114847]) (Figure 1 and the online data supplement, Table I) were designed using Ambion online tools (http://www.ambion. com). In addition, an siRNA directed against exon 10 of Best-3 but with 2 mismatching nucleotides (mutated siRNA) was designed. This mutated siRNA lost its effect against Best-3 and was used as the control for unspecific and microRNA effects of the transfection.28 A second control used in this study was nonrelated siRNA directed against enhanced green fluorescent protein (Ambion Ltd).
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Transfection of Cell Culture
Cultured rat aortic smooth muscle cells (A7r5) were transfected with siRNA using the TransIT TKO transfection kit (Mirus Bio Co) following the protocol of the manufacturer. The cells were used 72 hours later for quantitative PCR (qPCR), Western blot analysis, or patch-clamp studies.
Transfection of Intact Arteries
All experiments were conducted in accordance with national guidelines for animal research. Second and third order branches (internal diameter, 
250 µm) of the mesenteric artery were transfected in vivo with siRNA. Rats were euthanized 3 days after operation and the relevant arteries were isolated and used for qPCR, Western blot analysis, and patch-clamp studies.
Blood Vessels
Male Wistar rats, 12 to 18 weeks old, were euthanized with CO2 inhalation. Second and third order branches of the mesenteric artery, pulmonary arteries, and aorta were dissected out in ice-cold PBS. Arterial segments were used for PCR, Western blot, and smooth muscle cell (SMC) isolation.
RT-PCR
The expression of Best-1, Best-2, and Best-3 was investigated by RT-PCR (accession numbers and primer sequences in supplemental Table I). The expression of GAPDH gene was used as a control. The specificity of the reaction (for Best-3) was confirmed by sequencing (MWG-Biotech AG) the product that had the expected size.
RNA Quantification
The primers for qPCR (Figure 1 and supplemental Table I) were chosen to produce amplified regions spanning an intron in the genomic sequence, as well as the target sequences for siRNA. qPCR was performed using a SYBR Green assay. Using the computer-indicated fluorescence threshold, the knockdown was calculated with reference to nontransfected control.29 GAPDH was used for load control.
Western Blot
Antibodies against Best-3 protein, together with immunizing peptide, was provided by FabGennix Inc and characterized by expression of the epitope (supplemental Figure I). Protein from lysed arteries and confluent A7r5 was detected by standard Western blot protocol and quantified using ImageJ program (version 1.37, NIH). The detected protein was expressed relative to total protein. Preabsorption using the immunizing peptide abolished staining in all cases (supplemental Figure I).
Single SMC Staining
The semiquantitative expression level of Best-3 protein in SMCs from in vivo–transfected arteries was detected. SMCs isolated1 from the arteries were fixed and incubated overnight with the primary antibody, which was detected next day with Alexa-488 fluorescent-conjugated secondary antibody using a confocal microscope (LSM-5 Pascal Exciter, Zeiss, Germany; emission/excitation at 530/488 nm). Detected protein was quantified using ImageJ program (version 1.37, NIH) as a ratio of intensity between transfected and corresponding nontransfected cells.
Patch-Clamp Recording
Recordings were made in whole-cell configuration. Unless otherwise stated, Ca2+-activated currents were evoked by pipette solutions containing calcium buffered at 900 nmol/L, as described previously.1,3 The membrane conductances and currents were normalized to cell capacitances.
Data Analyses
All data are given as means±SEM. For RNA and protein detection, n is the number of independent experiments made on different batches of cells or different animals. For patch-clamp results, n is the number of recorded cells from at least 3 different batches of cells. Student’s t test was used for single comparisons, and 1-way ANOVA with Bonferroni’s post test was used for multiple comparisons (version 2.01, GraphPad Software). Values of P<0.05 were considered statistically significant.
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Results |
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We have previously reported3 a cGMP-dependent, Ca2+-activated Cl– current with a linear current–voltage relationship in smooth muscle cells (SMCs) isolated from rat mesenteric small arteries and aorta that is not present in SMCs from pulmonary arteries (Figure 2A). Here, we show that also the rat aortic SMC line A7r5 expresses this current (Figures 2A and 3
A). Similar to SMCs isolated from the vasculature, cultured A7r5 cells expressed 2 types of Ca2+-activated Cl– conductances that differed in their biophysical and pharmacological characteristics.3
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Ca2+-activated current was initially evoked by cell dialysis with high Ca2+ solution (free [Ca2+]i 900 nmol/L, 48 cells in total). This current was significantly inhibited by 100 µmol/L niflumic acid (NFA) (n=15; Figure 3A) but not by 10 µmol/L Zn2+ (supplemental Figure II). The inhibitory effect of NFA was not mimicked by the vehicle (0.1% ethanol, n=6; data not shown). When intracellular Ca2+ was nominally 0, no NFA-sensitive current was seen (n=8, supplemental Figure III). Changes in Cl– concentration revealed that the reversal potential of the NFA-sensitive current followed the equilibrium potential for Cl–, indicating that this current is carried by Cl– (n=8; supplemental Figure IV). This NFA-sensitive current in A7r5 cells has a voltage dependence and Ca2+ sensitivity similar to the classic Ca2+-activated Cl– current shown in freshly isolated vascular SMCs.30,31 The NFA-sensitive Cl– current rectified in the outward direction at low [Ca2+]i (30 nmol/L), whereas its rectification diminished at higher [Ca2+]i (900 nmol/L) (supplemental Figure III). This Ca2+-dependent rectification is known for the classic Ca2+-activated Cl– conductances.30,31
In the continuous presence of 100 µmol/L NFA, 300 µmol/L the membrane-permeable cGMP analog 8Br-cGMP activated another conductance that was sensitive to 10 µmol/L Zn2+ (n=15; Figure 3A). This cGMP-dependent conductance was also Ca2+-sensitive (supplemental Figure III). The cGMP-dependent Ca2+-activated current was voltage-independent through the entire [Ca2+]i range. The competitive protein kinase G (PKG) antagonist Rp-8Br-PET-cGMPS prevented this current (n=6; supplemental Figure V), as did the membrane-permeable PKG inhibitor peptide DT-2 (n=6; supplemental Figure V), strongly suggesting an involvement of PKG-dependent phosphorylation in its activation. The amplitude of the NFA-sensitive current was not affected by PKG inhibition (n=6; supplemental Figure V). Cl– substitutions revealed that the cGMP-dependent Ca2+-activated current was a Cl– current (n=8; supplemental Figure IV). Thus, in cultured A7r5 cells, this current has characteristics similar to the cGMP-dependent Cl– current described previously in SMCs from rat mesenteric small arteries,1,2 several other vascular beds, and colon.3 In accordance with the previous findings,2,3 10 µmol/L Zn2+ inhibited the cGMP-dependent Cl– current (Figure 3A) and 8Br-cGMP could not elicit the current in the presence of 10 µmol/L Zn2+ (n=4; supplemental Figure II). Thus, A7r5 cells express at least 2 different types of Ca2+-activated Cl– currents of similar magnitude, 1 of which is the cGMP-dependent Ca2+-activated Cl– current (Figure 4).
Using RT-PCR with gene-specific primers, we identified mRNA expression of the Best-3 gene in rat mesenteric small arteries, rat aorta, and A7r5 cells, whereas only very low expression was seen in rat pulmonary arteries (n=4; Figure 2B). A weak expression of Best-1 and Best-2 genes was also seen (n=3, data not shown). Using an antibody against the Best-3 band with the expected molecular weight was detected in lysates from rat mesenteric small arteries, aorta, and A7r5 cells (n=3; Figure 2C). Only 1 band (65 kDa) was detected, and it was not seen when preincubated with the immunizing peptide (supplemental Figure I). In contrast, the Best-3 protein could not be detected in rat pulmonary arteries (Figure 2C), consistent with our mRNA data. This distribution was similar to the distribution of the cGMP-dependent Ca2+-activated Cl– conductance in SMCs. It is relevant in this respect that mouse Best-3 protein was recently suggested to be under strong inhibitory influence of its C terminus,24 which might be regulated by PKG.25 Based on the distribution pattern, together with potential PKG sensitivity, we focused our study on a functional role of endogenous Best-3 in vascular smooth muscles. Because of lack of specific pharmacological tools a siRNA approach was applied.
Separate batches of confluent A7r5 cells were transfected with 2 different Best-3–directed siRNAs, mutated siRNA or nonrelated siRNA. Nearly 100% of the cells received siRNA after transfection, as judged by fluorescence of Cy3-labeled siRNA (Figure 5A). Transfection with Best-3–directed siRNAs (exons 7 and 10) resulted in a significant downregulation of the level of Best-3 mRNA (Figure 5B and 5C). A7r5 cells transfected with the nonrelated siRNA or cells that were transfected with mutated siRNA had similar expression of the Best-3 gene as nontransfected controls (Figure 5C). Transfection with siRNAs did not affect Best-1 and Best-2 isoforms. Best-1 mRNA was 104±4% (n=3) of control in cells transfected with nonrelated siRNA and 102±9% (n=3) in cells transfected with Best-3–directed siRNA (exon 10). Best-2 mRNA was 94±14% (n=3) and 98±18% (n=3), respectively. The downregulation of the Best-3 mRNA level was associated with a significant downregulation of Best-3 protein expression (Figure 5D and 5E). When cells were transfected with nonrelated siRNA, the Best-3 protein level was not different from the nontransfected control.
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Transfection of A7r5 did not affect the initial (recording 1, Figure 3) or the background currents (recording 4, Figure 3). The initial current amplitudes at –60 mV were –22.8±3.4 pA/pF in nontransfected cells (n=15, 26.2±4.5 pF) and –20.8±3.9 pA/pF in Best-3–siRNA (exon 10) transfected cells (n=12, 24.5±3.1 pF); the background currents were –14.1±2.9 pA/pF and –13.9±3.9 pA/pF, respectively. Transfection with the Best-3–directed siRNA (exon 10) significantly suppressed the cGMP-dependent Ca2+-activated Cl– current (n=12; Figure 3B), whereas nonrelated siRNA transfection (n=15) had no effect on the Cl– current in comparison with nontransfected cells (n=6; Figure 4). In another experimental series, A7r5 cells transfected with siRNA directed against exon 7 of the Best-3 gene were compared with cells transfected with mutated Best-3 siRNA and nonrelated siRNA, as well as with nontransfected control (Figure 6). Only cells transfected with the Best-3–directed siRNA showed significant suppression of the cGMP-dependent Ca2+-activated Cl– conductance (P<0.001). The normalized whole-cell cGMP-dependent conductances were 63±17 pS/pF (cell membrane capacitance [Cm]=24±4 pF, n=9) in nontransfected cells, 10±2 pS/pF (Cm=35±8 pF, n=8) in cells transfected with Best-3–directed siRNA (exon 7), 69±17 pS/pF (Cm=28±5 pF, n=8) in cells transfected with nonrelated siRNA, and 51±7 pS/pF (Cm=39±11 pF, n=8) in cells transfected with mutated siRNA. The NFA-sensitive, Ca2+-activated Cl– conductance had the same magnitude in all groups of cells; 68±20 pS/pF, 65±18 pS/pF, 77±17 pS/pF, and 73±10 pS/pF, respectively. Thus, transfection of A7r5 cells with the Best-3–directed siRNAs specifically suppressed Best-3 mRNA (Figure 5C), protein (Figure 5E), and the cGMP-dependent Ca2+-activated Cl– current (Figures 3, 4, and 6
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Although the Best-3–directed siRNA effectively and specifically suppressed the native cGMP-dependent Ca2+-activated Cl– current in cultured cells, the question remained whether this conductance is associated with Best-3 expression in intact vessels. To address this issue, we transfected in vivo a segment of rat mesenteric small artery with either Best-3–directed (exon 10) or nonrelated siRNAs. Three days later, we detected significant suppression of expression of both Best-3 mRNA and protein in arteries transfected with the Best-3–directed siRNA, whereas no changes were seen after transfection with nonrelated siRNA (Figure 7). Similar to the results obtained on A7r5 cells (Figures 4 through 6), the reduction of Best-3 protein expression was associated with suppression of the cGMP-dependent Ca2+-activated Cl– current without an effect on the magnitude of the NFA-sensitive, Ca2+-activated Cl– current (Figure 7 and supplemental Figure VI).
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Discussion |
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A cGMP-dependent Ca2+-activated Cl– current with unique characteristics was recently described in mesenteric small artery SMCs by our group1 and by Piper and Large.2 This current has a similar magnitude as the classic Ca2+-activated Cl– conductance in SMCs from mesenteric arteries, suggesting its importance for vascular function.3 Although the cGMP-dependent Ca2+-activated Cl– current was characterized in detail electrophysiologically,1,2 its molecular identity has been unknown. In the present study, we identified a protein necessary for this Cl– conductance.
We have previously reported that the cGMP-dependent Ca2+-activated Cl– current is present in SMCs from most vascular beds,3 with the notable exception of the pulmonary arteries (Figure 2). This current is distinct from previously described cGMP-independent Ca2+-activated Cl– currents in that it is voltage-independent and highly sensitive to Zn2+.1,2 The cGMP-independent Cl– currents, in contrast, showed increasing outward rectification with reduction of [Ca2+]i and greater sensitivity to NFA than the cGMP-dependent current.4,5 Based on the voltage dependence, the sensitivity to blockers and intracellular Ca2+ and activation by cGMP analogs, we report here that both the classic cGMP-independent and the novel cGMP-dependent Ca2+-activated Cl– currents coexist in a SMC line (A7r5).
The identification of the protein for the Ca2+-activated Cl– current faces several problems, eg, the absence of selective blockers and expression of endogenous Ca2+-activated Cl– currents in most cell lines, which makes interpretation of currents induced by heterologous expression of gene candidates difficult.5,7 Recently, bestrophins, the gene products of four mammalian Best genes,15,32 were suggested to encode Ca2+-activated Cl– channels based on heterologous expression9,10,16 and siRNA experiments.18,19,27
We hypothesized that a product of the Best gene family could be responsible for the cGMP-dependent Ca2+-activated Cl– current based on similarities between the membrane current produced by expression of different bestrophin isoforms9,10,16,17 and the cGMP-dependent Ca2+-activated Cl– current.1–3 Both currents were shown to express a linear current–voltage relationship1,2,10,25 and to have a high sensitivity (100 to 200 nmol/L) to intracellular calcium.2,10 Furthermore, the GABA channel M2 pore domain responsible for high sensitivity to Zn2+26 was previously shown to have similar structure and physical properties as the putative RFP pore domain of bestrophins.10 This similarity could suggest that the bestrophins share sensitivity to Zn2+ with the GABA channel.
A second line of evidence that a member of the Best family is responsible for the cGMP-dependent Ca2+-activated Cl– current comes from similarities in distribution patterns for Best-3 gene expression. In this study, we show that Best-3 is expressed both at the mRNA level and at the protein level in rat mesenteric small arteries, rat aorta, and A7r5 cells but not in pulmonary arteries. The same distribution of the cGMP-dependent Ca2+-activated Cl– current has been described recently,3 as well as in the present study.
The key observation of the present study is that downregulation of Best-3 mRNA and protein is associated with a significant reduction of the cGMP-dependent Ca2+-activated Cl– current, both in cultured cells and in vascular SMCs in vivo. In this respect, it is important that the magnitudes of the NFA-sensitive Ca2+-activated Cl– current and the background current were not affected. This is consistent with a specific effect of the siRNA directed against Best-3 gene. The specificity of siRNA action was further confirmed in accordance with the strategy recently suggested.28 Two siRNAs directed against different exons of the Best-3 gene were applied at minimally effective concentration and similar efficiencies with respect to Best-3 mRNA, and the cGMP-dependent current were demonstrated. We cannot completely exclude off-target effects of siRNA that are not homologous with the target genes.28 However, the related genes Best-1 and Best-2 were not affected. Furthermore, the absence of any changes after transfection with nonrelated siRNA or the mutated Best-3 siRNA suggests that our approach has a high degree of specificity. We found that 45% of Best-3 protein remained in cells and arteries transfected against Best-3 gene, whereas the Best-3 mRNA was reduced to 20% and the cGMP-dependent Ca2+-activated Cl– current was reduced even further. We have currently no explanation for this apparent discrepancy in magnitude of the reduction, but, because the antibody epitope is unique for Best-3 and we have detected only 1 band in the Western blot, we think it is unlikely to be attributable to unspecific binding. Interestingly, in a colonic epithelial cell line siRNA directed against Best-1 appears to reduce current more than protein19 consistent with our findings. Several groups have recently used siRNA directed against Best-1 to successfully downregulate a Cl– conductance in cultured cells.18,21,27 Finally, Kunzelmann and colleagues19 suggested a direct association between Best-1 expression and native Ca2+-activated Cl– currents in epithelial cells of different organs.
The parallel reduction of the cGMP-dependent Ca2+-activated Cl– current and Best-3 expression does not provide information on whether Best-3 protein forms the channel pore or functions as a channel modulator. Nevertheless, the membrane current carried by heterologously expressed bestrophins was previously shown to be inhibited by a modification of sulfhydryl groups and by point mutations within the hypothetical channel pore, which was interpreted to suggest a possible involvement of this protein in channel pore formation.15–18
The Best-3 associated current has a cGMP-dependence which is mediated via PKG activation (elsewhere1, 2 and the present study). Using a group-based phosphorylation scoring method,33 we identified in the predicted Best-3 protein several potential phosphorylation sites for PKG located preferably at the intracellular C-terminal (supplemental Figure VII). This finding is in accordance with the previous suggestion that the C-terminal cytoplasmic region contains several phosphorylation sites for protein kinases.25,27 Interestingly, it was shown recently that heterologous expression of the mouse Best-3 gene does not induce a Cl– conductance,24 whereas a large Cl– current was recorded after deletion of the C terminus, suggesting an inhibitory function of the C terminus. Thus, PKG-dependent phosphorylation might be an endogenous trigger that relieves this inhibition. It was also suggested that this mechanism might be characteristic for all members of the bestrophin family.25 Furthermore, the epithelial Ca2+-activated Cl– conductance associated with Best-1 was recently suggested to be activated by NO via a cGMP-dependent pathway.20 Together with the presence of a possible PKG phosphorylation site in the C terminus, this is consistent with the near-absolute requirement for PKG-mediated phosphorylation of the native Cl– current studied here.1,2 Although the heterologously expressed human Best-3 protein produced large Cl– currents in excised membrane patches, the involvement of second messengers could not be excluded.34
The suggestion that bestrophins are a family of Ca2+-activated Cl– channels rests primarily on studies using heterologous expression9,10,15–17,24,25 and cell culture studies.19–21 As previously suggested, it may be difficult to couple a particular Cl– channel gene candidate to a physiological current using only this approach.5,7 Recent studies18,19,21,27 demonstrating that suppression of Best-1 expression leads to downregulation of a Ca2+ activated Cl– current are in line with the present study, which provides strong novel evidence that another family member, Best-3, is responsible for an endogenous current and thus supports the notion that the Best gene family codes for Cl– channels. The present study, for the first time, demonstrates a direct coupling between endogenous Best-3 expression in vascular tissues and the appearance of the cGMP-dependent Ca2+-activated Cl– current. This suggests that the Best-3 gene is expressed in SMCs in vivo and that this expression is important for their membrane potential regulation1,35 by coupling intracellular calcium and NO/cGMP pathways. Because the bestrophin channels are generally accepted to be multimeric structures of the same or different bestrophins,9,14 the importance of other isoforms for this Ca2+-activated Cl– conductance in SMCs cannot be excluded and requires further investigation.
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Acknowledgments |
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We thank Susie Mogensen for excellent technical assistance.
Sources of Funding
This work was supported by the Danish Research Council, the Danish Heart Foundation, the Swedish Research Council, and the European Union Sixth Framework Programme ("RIGHT" program). The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Both authors contributed equally to this work.
Original received October 18, 2006; first resubmission received July 5, 2007; second resubmission received; April 29, 2008; revised second resubmission received July 16, 2008; accepted August 27, 2008.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Matchkov VV, Aalkjaer C, Nilsson H. A cyclic GMP-dependent calcium-activated chloride current in smooth-muscle cells from rat mesenteric resistance arteries. J Gen Physiol. 2004; 123: 121–134.
2. Piper AS, Large WA. Single cGMP-activated Ca2+-dependent Cl– channels in rat mesenteric artery smooth muscle cells. J Physiol. 2004; 555: 397–408.
3. Matchkov VV, Aalkjaer C, Nilsson H. Distribution of cGMP-dependent and cGMP-independent Ca2+-activated Cl– conductances in smooth muscle cells from different vascular beds and colon. Pflugers Arch. 2005; 451: 371–379.[CrossRef][Medline] [Order article via Infotrieve]
4. Large WA, Wang Q. Characteristics and physiological role of the Ca2+-activated Cl– conductance in smooth muscle. Am J Physiol. 1996; 271: C435–C454.[Medline] [Order article via Infotrieve]
5. Hartzell C, Putzier I, Arreola J. Calcium-activated chloride channels. Annu Rev Physiol. 2005; 67: 719–758.[CrossRef][Medline] [Order article via Infotrieve]
6. Leblanc N, Ledoux J, Saleh S, Sanguinetti A, Angermann J, O'Driscoll K, Britton F, Perrino BA, Greenwood IA. Regulation of calcium-activated chloride channels in smooth muscle cells: a complex picture is emerging. Can J Physiol Pharmacol. 2005; 83: 541–556.[CrossRef][Medline] [Order article via Infotrieve]
7. Pusch M. Ca2+-activated chloride channels go molecular. J Gen Physiol. 2004; 123: 323–325.
8. Petrukhin K, Koisti MJ, Bakall B, Li W, Xie G, Marknell T, Sandgren O, Forsman K, Holmgren G, Andreasson S, Vujic M, Bergen AA, McGarty-Dugan V, Figueroa D, Austin CP, Metzker ML, Caskey CT, Wadelius C. Identification of the gene responsible for Best macular dystrophy. Nat Genet. 1998; 19: 241–247.[CrossRef][Medline] [Order article via Infotrieve]
9. Sun H, Tsunenari T, Yau KW, Nathans J. The vitelliform macular dystrophy protein defines a new family of chloride channels. Proc Natl Acad Sci U S A. 2002; 99: 4008–4013.
10. Qu Z, Wei RW, Mann W, Hartzell HC. Two bestrophins cloned from Xenopus laevis Oocytes express Ca2+-activated Cl– currents. J Biol Chem. 2003; 278: 49563–49572.
11. Bakall B, Marknell T, Ingvast S, Koisti MJ, Sandgren O, Li W, Bergen AA, Andreasson S, Rosenberg T, Petrukhin K, Wadelius C. The mutation spectrum of the bestrophin protein-functional implications. Hum Genet. 1999; 104: 383–389.[CrossRef][Medline] [Order article via Infotrieve]
12. Milenkovic VM, Langmann T, Schreiber R, Kunzelmann K, Weber BH. Molecular evolution and functional divergence of the bestrophin protein family. BMC Evol Biol. 2008; 8: 72.[CrossRef][Medline] [Order article via Infotrieve]
13. Kunzelmann K, Milenkovic VM, Spitzner M, Soria RB, Schreiber R. Calcium-dependent chloride conductance in epithelia: is there a contribution by Bestrophin? Pflugers Arch. 2007; 454: 879–889.[CrossRef][Medline] [Order article via Infotrieve]
14. Hartzell HC, Qu Z, Yu K, Xiao Q, Chien LT. Molecular physiology of bestrophins: multifunctional membrane proteins linked to best disease and other retinopathies. Physiol Rev. 2008; 88: 639–672.
15. Tsunenari T, Sun H, Williams J, Cahill H, Smallwood P, Yau KW, Nathans J. Structure-function analysis of the bestrophin family of anion channels. J Biol Chem. 2003; 278: 41114–41125.
16. Qu Z, Fischmeister R, Hartzell C. Mouse bestrophin-2 is a bona fide Cl– channel: identification of a residue important in anion binding and conduction. J Gen Physiol. 2004; 123: 327–340.
17. Qu Z, Hartzell C. Determinants of anion permeation in the second transmembrane domain of the mouse bestrophin-2 chloride channel. J Gen Physiol. 2004; 124: 371–382.
18. Chien LT, Zhang ZR, Hartzell HC. Single Cl– channels activated by Ca2+ in Drosophila s2 cells are mediated by bestrophins. J Gen Physiol. 2006; 128: 247–259.
19. Barro Soria R, Spitzner M, Schreiber R, Kunzelmann K. Bestrophin 1 enables Ca2+ activated Cl– conductance in epithelia. J Biol Chem. In press.
20. Duta V, Duta F, Puttagunta L, Befus AD, Duszyk M. Regulation of basolateral Cl(-) channels in airway epithelial cells: the role of nitric oxide. J Membr Biol. 2006; 213: 165–174.[CrossRef][Medline] [Order article via Infotrieve]
21. Spitzner M, Martins JR, Soria RB, Ousingsawat J, Scheidt K, Schreiber R, Kunzelmann K. Eag1 and Bestrophin 1 are upregulated in fast growing colonic cancer cells. J Biol Chem. 2008; 283: 7421–7428.
22. Rosenthal R, Bakall B, Kinnick T, Peachey N, Wimmers S, Wadelius C, Marmorstein A, Strauss O. Expression of bestrophin-1, the product of the VMD2 gene, modulates voltage-dependent Ca2+ channels in retinal pigment epithelial cells. FASEB J. 2006; 20: 178–180.
23. Yu K, Xiao Q, Cui G, Lee A, Hartzell HC. The best disease-linked Cl– channel hBest1 regulates Ca V 1 (L-type) Ca2+ channels via src-homology-binding domains. J Neurosci. 2008; 28: 5660–5670.
24. Qu Z, Cui Y, Hartzell C. A short motif in the C-terminus of mouse bestrophin 4 inhibits its activation as a Cl channel. FEBS Lett. 2006; 580: 2141–2146.[CrossRef][Medline] [Order article via Infotrieve]
25. Qu ZQ, Yu K, Cui YY, Ying C, Hartzell C. Activation of Bestrophin Cl– channels is regulated by C-terminal domains. J Biol Chem. 2007; 282: 17460–17467.
26. Hosie AM, Dunne EL, Harvey RJ, Smart TG. Zinc-mediated inhibition of GABA(A) receptors: discrete binding sites underlie subtype specificity. Nat Neurosci. 2003; 6: 362–369.[CrossRef][Medline] [Order article via Infotrieve]
27. Duta V, Szkotak AJ, Nahirney D, Duszyk M. The role of bestrophin in airway epithelial ion transport. FEBS Lett. 2004; 577: 551–554.[CrossRef][Medline] [Order article via Infotrieve]
28. Cullen BR. Enhancing and confirming the specificity of RNAi experiments. Nat Methods. 2006; 3: 677–681.[CrossRef][Medline] [Order article via Infotrieve]
29. Tuzmen S, Kiefer J, Mousses S. Validation of short interfering RNA knockdowns by quantitative real-time PCR. Methods Mol Biol. 2007; 353: 177–203.[Medline] [Order article via Infotrieve]
30. Greenwood IA, Ledoux J, Leblanc N. Differential regulation of Ca2+-activated Cl– currents in rabbit arterial and portal vein smooth muscle cells by Ca2+-calmodulin dependent kinase. J Physiol. 2001; 534: 395–408.
31. Ledoux J, Greenwood I, Villeneuve LR, Leblanc N. Modulation of Ca2+-dependent Cl– channels by calcineurin in rabbit coronary arterial myocytes. J Physiol. 2003; 552: 701–714.
32. Kramer F, Stohr H, Weber BH. Cloning and characterization of the murine Vmd2 RFP-TM gene family. Cytogenet Genome Res. 2004; 105: 107–114.[CrossRef][Medline] [Order article via Infotrieve]
33. Xue Y, Zhou F, Zhu M, Ahmed K, Chen G, Yao X. GPS: a comprehensive www server for phosphorylation sites prediction. Nucleic Acids Res. 2005; 33: W184–W187.
34. Tsunenari T, Nathans J, Yau KW. Ca2+-activated Cl– current from human bestrophin-4 in excised membrane patches. J Gen Physiol. 2006; 127: 749–754.
35. Peng H, Matchkov V, Ivarsen A, Aalkjaer C, Nilsson H. Hypothesis for the initiation of vasomotion. Circ Res. 2001; 88: 810–815.
36. Grunweller A, Gillen C, Erdmann VA, Kurreck J. Cellular uptake and localization of a Cy3-labeled siRNA specific for the serine/threonine kinase Pim-1. Oligonucleotides. 2003; 13: 345–352.[CrossRef][Medline] [Order article via Infotrieve]
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