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Originally published In Press as doi:10.1074/jbc.M308414200 on August 25, 2003

J. Biol. Chem., Vol. 278, Issue 49, 49563-49572, December 5, 2003
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Two Bestrophins Cloned from Xenopus laevis Oocytes Express Ca2+-activated Cl- Currents*

Zhiqiang Qu, Raymond W. Wei, Wesley Mann, and H. Criss Hartzell{ddagger}

From the Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, July 31, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+-activated Cl- channels play important diverse roles from fast block to polyspermy to olfactory transduction, but their molecular identity has not been firmly established. By searching sequence databases with the M2 pore domain of ligand-gated anion channels, we identified potential Ca2+-activated Cl- channels, which included members of the bestrophin family. We cloned two bestrophins from Xenopus oocytes, which express high levels of Ca2+-activated Cl- channels. The Xenopus bestrophins were expressed in a variety of tissues. We predict that bestrophin has six transmembrane domains with the conserved RFP domain playing an integral part in ionic selectivity. When Xenopus bestrophins were heterologously expressed in human embryonic kidney-293 cells, large Ca2+-activated Cl- currents were observed. The currents are voltage- and time-independent, do not rectify, have a Kd for Ca2+ of ~210 nM, and exhibit a permeability ratio of I- > Br- > Cl- >> aspartate. The W93C and G299E mutations produce non-functional channels that exert a dominant negative effect on wild type channels. We conclude that bestrophins are the first molecularly identified Cl- channels that are dependent on intracellular Ca2+ in a physiological range.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+-activated Cl- currents play a variety of important physiological roles that include functions as diverse as the fast block to polyspermy and olfactory transduction (1). Despite their physiological importance, the molecular identity of these channels remains in question. The CLCA family, initially cloned from bovine trachea by Cunningham et al. (2), has been proposed to code for Ca2+-activated Cl- channels. Heterologous expression of hCLCA1, hCLCA2, mCLCA1, mCLCA2, bCLCA1, and pCLCA1 results in an increased Cl- current that is stimulated by intracellular Ca2+ (3-7). However, CLCA proteins have not been universally accepted as Ca2+-activated Cl- channels for several reasons (8, 9). The properties of CLCA-induced currents differ depending on the cell type in which they are expressed. Furthermore, some CLCAs are cell adhesion proteins or secreted proteins with poorly conserved trans-membrane architecture. Furthermore, the current is activated by very high, non-physiological Ca2+ concentrations, and native expression of CLCAs does not correlate with Ca2+-activated Cl- currents.

Several years ago, we began an in silico approach to identify novel Cl- channel genes. Our previous studies on the endogenous Ca2+-activated Cl- channels of Xenopus oocytes suggested that these channels might have a pore architecture similar to ligand gated anion channels (LGACs)1 of the GABA(A), GABA(C), and glycine receptor family (10, 11). LGACs and Ca2+-activated Cl- channels have similar anion selectivity sequences and similar pore dimensions. Furthermore, both are blocked by anion channel blockers that enter the pore selectively from the extracellular side, unlike cystic fibrosis transmembrane conductance regulator and ClC channels that seem to have the wide end of their pores oriented cytoplasmically (12).

The determinants of ligand-gated channel anion selectivity have been well studied. Mutagenesis of both anion- and cation-selective ligand-gated channels have established that three rings of charged residues formed by the M2 transmembrane domains of the channel subunits play a major role in ion selectivity (13, 14). In LGACs, both the intermediate and extracellular rings are positively charged, whereas in cation-selective channels these residues are negatively charged. Although both the extracellular (15) and intermediate rings (16) contribute to ion selectivity, the intermediate ring is critical, because the pore tapers to a constriction at this point to bring the charged residues of the intermediate ring close to the permeant ion. This geometric constraint is apparently conferred on the channel by a proline residue at position -2' (17, 18). Mutations affecting the proline and the positively charged intermediate ring can convert selectivity from anionic to cationic (17, 18).

We used degenerate M2 domains of LGACs to search for potential unique Cl- channels. Among the hits we obtained was a family of proteins called bestrophins. Human bestrophin-1 (VMD2) was positionally cloned in 1998 from a Swedish family with an inherited form of early onset macular degeneration called Best vitelliform macular dystrophy (19). Bestrophin was initially thought to be involved in fatty acid transport (19), but more recently (20), it has been shown that human bestrophin-1 expressed in heterologous systems induces a chloride current. The possibility that bestrophin is a Cl- channel is particularly exciting, because the hallmark diagnostic feature of Best disease is an abnormal electro-oculogram (21, 22). The slow light peak of the electro-oculogram, which is reduced in Best disease, is thought to reflect an increase in Cl- conductance across the basolateral membrane of the retinal pigment epithelium (RPE) (23). Trans-RPE transport plays an important role in maintaining the fluid and ionic composition of the fluid surrounding the photoreceptors. Recently (24), it has been demonstrated that human bestrophin-1 is localized to the basolateral membrane of RPE, making it a candidate for the basolateral Cl- channel of RPE.

In this paper, we report the cloning and expression of two bestrophins from Xenopus oocyte mRNA. These proteins have high similarity to human bestrophins and induce Cl- currents when expressed in heterologous systems.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning—Expressed sequence tags for Xenopus bestrophins were identified by BLAST searches using conserved domains of mammalian bestrophins. PCR primers were constructed, and full-length Xenopus bestrophins were obtained by RT-PCR using total RNA isolated from Xenopus oocytes. The resulting PCR products were subcloned into TOPO-PCRII and sequenced in both forward and reverse directions. Each clone was sequenced at least three times. xBest-2b was subcloned into pIRES2-EGFP (Invitrogen) by PCR. The forward PCR primer contained a BglII restriction site, and the reverse primer contained a SacII restriction site for insertion into the pIRES2-EGFP vector.

Site-specific Mutation of xBest-2a—Site-specific mutations were made using a PCR-based site-directed mutagenesis kit (QuikChange; Stratagene). Specific mutations were introduced into primers. The template, xBest-2a in pCMV-Sport6, was amplified with the Pfu DNA polymerase by the polymerase chain reaction. The methylated original templates were digested with DpnI, and the PCR products were transformed into XL-1 Blue Escherichia coli. Mutations were confirmed by DNA sequencing.

Heterologous Expression of Bestrophins in HEK-293 Cells—xBest-2a subcloned in pCMV-Sport6 was transfected into HEK-293 cells with pIRES2-EGFP vector at a ratio of 10:1. xBest-2b was subcloned in pIRES2-EGFP vector. Plasmid transfection was carried out with Fu-GENE 6 kit (Roche Applied Science) or Ca2+-PO4 precipitation (Clontech). Transfected cells were dissociated and replated 1 day after transfection and spread on glass coverslips. Fluorescent cells were used for patch clamp experiments within 3 days.

Antibody Production—Antibodies were raised against a peptide of xBest-2a. The amino acid sequence was EFQSQEPIQDPPYN, which corresponds to amino acids 451-464 of xBest-2a. xBest-2b differs in six of these 14 residues (VFQFPETVQDPPNN). BLAST hits with an E value <10 included only xBest-2a. Peptides were synthesized by Research Genetics (Invitrogen) using multiple antigenic peptide resin technology to enhance antigenic response. The peptides (with a cysteine added to the C terminus) were conjugated to KLH (keyhole limpet hemocyanin), emulsified with an equal volume of Freud's incomplete adjuvant, and injected into two New Zealand White rabbits. The animals received a boost after two, six, and eight weeks. Sera were assayed by enzyme-linked immunosorbent assay and Western blot analysis with GST fusion proteins of bestrophins. The sera were then affinity-purified.

GST Fusion Proteins—GST fusion proteins of several bestrophins were engineered by subcloning the bestrophin C-terminal tail into pGEX-4T expression vectors (Amersham Biosciences) in-frame with GST. The C-terminal fragments used for xBest-2a and xBest-2b were residues 291-512 (GEQLIN... LSVAT). The resulting GST fusion protein consisted of N-terminal GST followed by the C-terminal fragments of the bestrophin. The fusion proteins were grown in BL21-Gold(DE3) competent E. coli (Stratagene) and induced by isopropyl-1-thio-{beta}-D-galactopyranoside at a final concentration of 1 mM for 3-4 h at 37 °C. Bacterial pellets were resuspended in 50 mM Tris, pH 8.0, 40 mM EDTA, 2.5% sucrose, 0.02% NaN3, 1 mM phenylmethylsulfonyl fluoride, 2.5 mg/ml lysozyme, and protease inhibitor mixture set II (5 µl/20 mg bacteria; Calbiochem). The lysate was subjected to French press twice at 1000 p.s.i. The lysate was centrifuged at 10,000 rpm for 15 min and then passed over a glutathione-Sepharose 4B column (Amersham Biosciences). The fusion proteins were eluted from the columns with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0.

Total RNA Isolation and Purification—Total RNA from Xenopus RPE, neural retina, brain, spleen, gut, lung, liver, blood, heart, and oocyte were isolated using Trizol reagent (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. Tissue (1 g/10 ml Trizol) was homogenized using a Polytron (Brinkman) homogenizer. The Trizol was extracted two times with chloroform, and the total RNA was recovered by precipitation with isopropyl alcohol. DNA was removed by DNase treatment and DNA-freeTM (Ambion).

Semi-quantitative PCR—cDNA was synthesized from total RNA using ThermoScriptTM reverse transcriptase (Invitrogen) primed with oligo(dT). The reaction mixture contained 4 µg of total RNA, 2.5 µM oligo(dT), 1 mM dNTP mix, 0.1 mM dithiothreitol, 2 units/µl RNaseOUT, and 0.75 units/µl ThermoScriptTM reverse transcriptase. The cDNA was amplified by using xBest-2a specific primers and SYBR Green JumpStartTM Taq ReadyMixTM (Sigma). The concentration of primers was 40 nM. Data were analyzed with iCycler software, which determines the threshold cycle for each sample. The house keeping gene GAPDH of the same cDNA sample was also amplified. The quantification of the xBest-2a gene is defined by the ratio of the threshold cycle values of xBest-2a against the GAPDH in the same sample. The thermocycler used for quantitative detection was iCycler (Bio-Rad), and the protocol was as follows: one cycle at 95 °C for 2 min, 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 30 s. The primers for xBest-2a were 5'-TTG GCT GAA GGT GGG TGA ACA-3' and 5'-GGG CGC GGG TCT GAG TGA TT-3'. The primers for GAPDH were 5'-GAC CTG CCG CCT GCA GAA G-3' and 5'-GAC TAG CAG GAT GGG CGA C-3'.

SDS-PAGE and Western Blots—Xenopus laevis were acutely decapitated, and organs were quickly collected and weighed. The organs were homogenized (50 mg tissue per ml) in the LSB buffer (60 mM NaCl, 25 mM Na-PIPES, pH 6.9, 1 mM EDTA, 2 mM NaN3, 0.3 mM {beta}-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 100-fold diluted protease inhibitor mixture III (Calbiochem)). SDS was added to a final concentration of 1%. DNA was sheared by passing the solution through a 20-gauge needle. Protein concentration was measured by the Bradford assay (Bio-Rad) and checked with Coomassie Blue staining in gels. Protein samples were run on 4-15% gradient polyacrylamide gels in 25 mM Tris-HCl, pH 8.3, 200 mM glycine, 0.5% SDS with ~10 µg of protein per well. The proteins were transferred electrophoretically to Hybond nitrocellulose membranes in 25 mM Tris-HCl, pH 8.3, 200 mM glycine, 20% methanol. The membranes were blocked with 5% dry milk in PBS-T (PBS with 0.1% Tween 20) overnight at 4 °C or 1 h at room temperature. The blot was incubated with primary antibody (1/1000 dilution) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1/7000) (Jackson ImmunoResearch Laboratories) in PBS-T with 1% dry milk. Immunoreactivity was visualized by enhanced chemiluminescence (ECL kit; Amersham Biosciences).

Immunocytochemistry—HEK-293 cells were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.3, for 2 h. The cells were washed three times with PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 6 mM NaH2PO4, pH 7.3) and then blocked with PBS containing 0.1% Triton X-100, 3% bovine serum albumin, and 10% normal goat serum for 30 min. The cells were then incubated at 4 °C overnight with 180 ng/ml affinity-purified A5925 in PBS containing 2% bovine serum albumin overnight. The cells were then washed extensively with PBS and incubated with Alexa-488 (Molecular Probes)-conjugated goat anti-rabbit IgG (Molecular Probes) diluted 1:200. The cells were visualized using a Zeiss 510 confocal microscope.

Electrophysiological Methods—Recordings were performed using the whole-cell recording configuration of the patch clamp technique. Patch pipettes were made of borosilicate glass (Sutter Instrument Co., Novato, CA), pulled by a Sutter P-2000 puller (Sutter Instrument Co.), and fire-polished. Patch pipettes had resistances of 3-5 megohms 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 gravity-feed of the 1-ml chamber at a speed of ~4 ml/min. To measure the steady-state current-voltage relationship, the cells were voltage-clamped from a holding potential of 0 mV with 700-ms-duration pulses from -100 to +100 mV. To measure the instantaneous current-voltage relationship, a pre-pulse was first applied to +100 mV for 200 ms and then 500-ms-duration voltage steps were applied between -100 and +100 mV. Data were acquired by an Axopatch 200A amplifier controlled by Clampex 8.1 via a Digidata 1322A data acquisition system (Axon Instruments, Foster City, CA). Experiments were conducted at room temperature (~24 °C).

Recording Solutions—The standard pipette solution contained the following (in mM): 146 CsCl, 2 MgCl2, 5 Ca2+-EGTA, 8 HEPES, 10 sucrose, pH 7.3. The "zero" free Ca2+ pipette solution contained 5 mM EGTA without added Ca2+ whereas high free Ca2+ pipette solution contained 5 mM Ca2+-EGTA made by the pH-metric method described by in Ref. 25. Working solutions having different free Ca2+ were prepared by mixing the zero-Ca2+ solution with the high Ca2+ solution in various ratios. The free [Ca2+] was calculated from the equation [Ca2+] = Kd x [Ca2+-EGTA]/[EGTA], where Kd is the Kd of EGTA (Kd = 1.0 x 10-7 M at 24 °C, pH 7.3, ionic strength 0.16 M). The calculated Ca2+ concentrations were confirmed in each solution by Fura-2 (Molecular Probes) measurements using an LS-50B luminescence spectrophotometer (PerkinElmer Life Sciences). In some experiments, pipette solution contained the following (in mM): 148 CsCl, 2 MgCl2, 0.5 CaCl2, 5 EGTA, 8 HEPES, pH 7.3, with free [Ca2+] = 164 nM.

The standard extracellular solution contained the following (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, 10 HEPES, pH 7.4. When Cl- was substituted with another anion, NaCl was replaced with NaX, where X is the substitute anion.

Solution osmolarity was 300 mosM for both intra- and extracellular solutions (Micro Osmometer, Model 3300; Advanced Instrument, Inc., Norword, MA). Slight differences in osmolarity were adjusted by addition of sucrose.

Analysis of Data—For the calculations and graphical presentation, we used Origin 6.0 software (Microcal, Northampton, MA). Relative halide permeability of the channels was determined by measuring the shift in Erev upon changing the bath solution from one containing 151 mM Cl- to another with 140 mM X and 11 mM Cl-, where X is the substitute anion. The permeability ratio was estimated using the Goldman-Hodgkin-Katz equation (26), PX/PCl = [Cl-]i/([X]o exp({Delta}ErevF/RT)) - [Cl-]o/[X]o, where {Delta}Erev is the difference between the reversal potential with the test anion X and that observed with symmetrical Cl-, and F, R, and T have their normal thermodynamic meanings.

Accession Numbers—xBest-2a has been entered in GenBankTM as accession number AY273825 [GenBank] , and xBest-2b has been entered as AY273826 [GenBank] . Other accession numbers are as follows: hBest1 (VMD2), NM_004183 [GenBank] ; hBest2 (VMD2L1), NM_017682 [GenBank] ; hBest3 (VMD2L2), NM_017682 [GenBank] ; hBest4 (VMD2L3), NM_152439 [GenBank] .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In Silico Cloning of Ca2+-activated Cl- Channels
BLAST searching (27) of protein data bases with degenerate M2 domains of LGACs found a number of proteins of the bestrophin family. Fig. 1 shows an alignment of the M2 domain of the human GABA(A) {alpha}1 subunit with four human bestrophins. The residues of the GABA(A) receptor are labeled in accordance with Keramidas (18) with the arginines of the intermediate and extracellular rings located at position 0' and 19', respectively. The homology between the bestrophins and the GABA(A) receptor in this region is rather weak (19% identity, 39% strong similarity). However, the critical residues for GABA(A) receptor ion selectivity are conserved: the 19' Rofthe extracellular ring, the 0' R of the intermediate ring, the -5' negative charge of the cytoplasmic ring, and the -2' proline at the pore constriction. The physical properties of the M2 domain of the human GABA(A)R and the RFP domain of xBest-2a are quite similar (Table I). Interestingly, the RFP sequence (positions 0 to -2') of the bestrophins that corresponds to the intermediate ring and associated proline of the GABA receptor is invariant among all 40+ members of the bestrophin family for which sequences are available, from Caenorhabditis elegans to human. This strongly suggests that the RFP sequence is crucial to channel function. We propose it is involved in ion selectivity.



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  		FIG. 1.
Comparison of human GABA(A) receptor {alpha}1 subunit M2 domain with the bestrophin RFP domain. The M2 domain of the human GABA(A){alpha}1 subunit is shown from residues 301-277. The intermediate positively charged ring at Arg-282 is labeled 0' (17). The outer positively charged ring is Arg-301 at position 19', and the negatively charged inner ring is Glu-277 at position-5'. The bestrophins are shown from residues 130-155. Identical residues are highlighted in red, similar residues in orange and weakly similar residues in blue.

 


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  		TABLE I
Physical properties of the M2 domain of GABA receptor and xBest-2a RFP domain

 
Because Ca2+-activated Cl- channels are expressed at high levels in Xenopus oocytes (12), we used the sequences obtained from mammalian bestrophins to find expressed sequence tags for Xenopus bestrophins. One Xenopus expressed sequence tag (BE669309 [GenBank] , Image clone 3401219) was obtained and sequenced and found to be a full-length bestrophin. The sequence was confirmed by sequencing full-length PCR products obtained by RT-PCR of mRNA isolated from Xenopus oocyte mRNA. At least three overlapping sequencing runs were performed. The PCR products from different clones differed reproducibly in sequence from one another, so new primers were designed to amplify a second, closely related transcript (xBest-2b). Fig. 2 compares the sequences of the two Xenopus bestrophins to four human bestrophins. Evolutionary relationships between 15 vertebrate bestrophins for which complete sequence information is available were determined by the neighbor joining method of Saitou and Nei (32) (Fig. 3A). The bestrophins from Zebrafish, Fugu, Xenopus, mouse, rat, and human fall into four groups with the both Xenopus bestrophins being in group 2. The two Xenopus bestrophins are 89% identical. Because X. laevis is pseudo-tetraploid, and xBest-2a and xBest-2b are more closely related to one another than any of the four human bestrophins are to one another, we believe that the Xenopus bestrophins are orthologs.



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  		FIG. 2.
Alignment of human and Xenopus bestrophins. Identical residues are shaded. Transmembrane domains predicted by the SOSUI algorithm (37) are indicated by horizontal lines. Consensus sequences for phosphorylation are highlighted in green for protein kinase A (PKA), red for protein kinase C (PKC), and blue for casein kinase II (casein). The signature RFP residues are cyan. The last 44 residues of hBest-1 are not shown. GenBankTM accession numbers are as follows: xBest-2a, AY273825 [GenBank] ; xBest-2b, AY2273826; hBest-1, NM_004183 [GenBank] ; hBest-2, NM_017682 [GenBank] ; hBest-3, NM_017682 [GenBank] ; hBest-4, NM_152439 [GenBank] .

 



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  		FIG. 3.
Properties of bestrophins. A, cladogram of vertebrate bestrophins. The relationships between Xenopus (x), human (h), mouse (m), Fugu (f), rat (r), and zebrafish (z) bestrophins were determined using the method of Saitou and Nei (32). Accession numbers are as in Fig. 2, except that Fugu sequences were obtained from the Fugu Genomics Project Assembly (fBest1 is SINFRUP00000141703, fBest2 is SINFRUP00000151123, and fBest-3 is SINFRUP00000134584). The Zebrafish bestrophins were cloned in our laboratory.2 B, hydropathy analysis of xBest-2a by the Kyte-Doolittle algorithm (38) with a window of 17 residues shows six distinct hydrophobic domains. This finding, coupled with the results of the SOSUI transmembrane prediction shown in Fig. 2, suggests six transmembrane domains. C, proposed topology of xBest-2a. Transmembrane domains are shaded yellow. Positively charged residues are red, negatively charged residues are blue, and proline is orange. Note the location of the RFP sequence at the cytoplasmic end of transmembrane helix 4.

 
All bestrophins are highly conserved in the first ~300 residues but differ significantly in the C-terminal third of the protein. xBest-2a is 95% identical to xBest-2b in the first 307 amino acids but only 85% identical in the remaining 205 amino acids. xBest-2a is 81% identical to hBest-2 in the first 307 amino acids but only 50% identical from residue 307 to the end.

The Xenopus bestrophins contain multiple protein kinase A, casein kinase, and protein kinase C phosphorylation consensus (PROSITE) sequences, but only three were conserved in the human proteins (Fig. 2). Thr-164 is a predicted casein kinase site that is conserved in all six bestrophins shown here and is conserved in a majority of the other bestrophins we have examined. Thr-6 and Thr-216 are predicted protein kinase C phosphorylation sites found in four and three, respectively, of the bestrophins shown here. The other phosphorylation sites were not conserved across species.

Kyte-Doolittle hydropathy analysis of xBest-2a with a window of 17 found six hydrophobic domains (Fig. 3B). The SOSUI algorithm predicted six transmembrane domains (shown as red lines over the sequence in Fig. 2). The (150)RFP sequence is at the C-terminal end of the 4th transmembrane domain and thus would be predicted to be located at the cytoplasmic mouth of the channel. Fig. 3C shows our model for xBest-2a bestrophin topology.

Bestrophin Expression
Semi-quantitative PCR—Real-time PCR was used to evaluate the tissue-specific expression of xBest-2. The primers that were chosen were identical to sequences in both xBest-2a and xBest-2b. Thus, the quantification represents the total of both orthologs (Fig. 4). xBest-2 mRNA is expressed at high levels in RPE, liver, and spleen and to a lesser extent in neural retina and lung. Relatively little message was found in brain, heart, and gut. Even though we were able to RT-PCR full-length xBest-2a and xBest-2b from RNA isolated from oocyte, we were unable to quantify the level of xBest-2 message by real-time PCR.



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  		FIG. 4.
Quantitative PCR. The amount of bestrophin-2 message in different Xenopus tissues was quantified by real-time PCR. The amount of bestrophin-2 message was compared with the amount of GAPDH message as described under "Experimental Procedures."

 
Western Blot Analysis—Polyclonal antibodies were developed to peptide sequences of the Xenopus bestrophins. Affinity-purified antibody A5925 was raised against residues 441-454 of xBest-2a (EFQSQEPIQDPPYN). This antibody specifically recognized GST fusion proteins of the C terminus of xBest-2a but not xBest-2b (Fig. 5A). Western blots with various Xenopus tissues showed that the A5925 antibody labeled a strong 54-kDa band in neural retina, liver, gut, and spleen (Fig. 5B). A faint 54-kDa band was also observed in brain, heart, lung, and oocyte. No band was observed in RPE. The 54-kDa band was absent when the antibody was pre-absorbed with the GST-xBest-2a fusion protein (Fig. 5C). The mass of xBest-2a predicted from the cDNA sequence is 59 kDa. We presume that the 54-kDa band corresponds to the predicted 59-kDa protein.



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  		FIG. 5.
Expression of bestrophin proteins in Xenopus tissues. A, specificity of antibody A5925. Different amounts of xBest-2b and xBest-2a GST fusion proteins (12-100 ng per well) were run on SDS-PAGE, blotted onto nitrocellulose, probed with affinity-purified A5925 antibody (90 ng/ml), and visualized with peroxidase-conjugated goat anti-rabbit secondary antibody by chemiluminescence. B, various Xenopus tissues were homogenized, dissolved in SDS buffer (see "Experimental Procedures."), run on SDS-PAGE, blotted onto nitrocellulose, and probed with affinity-purified A5925 antibody. C, a companion blot was probed with primary antibody (A5925) that had been incubated for 2 h with electrophoretically purified xBest-2a GST fusion protein. The blots in B and C were treated in parallel identically with the exception of the primary antibody absorption step. D, localization of xBest-2a in HEK-293 cells. xBest-2a in pCMV-SPORT6 was transfected into HEK-293 cells. 1-2 days after transfection, the cells were fixed, permeabilized, and stained with antibody A5925 against xBest-2a visualized with Alexa-488 conjugated second antibody (green). In the top row, cells were also stained with tetramethylrhodamine-conjugated WGA (red, left column), xBest-2 staining (green, middle column), and an overlay of the two stains (right column). In the bottom row, the cells were stained with A5925 against xBest-2a and a monoclonal mouse antibody against human transferrin receptor (TfR) followed by rhodamine goat anti-mouse secondary antibody. E, the distribution of xBest-2a at the membrane was determined by comparing red and green fluorescence along a line drawn across a cell. F, distribution of WGA- and xBest-2a-associated fluorescence along the line across the cell in E.

 
Expression in HEK Cells—xBest2a was expressed in HEK-293 cells by transient transfection. We observed two patterns of localization by immunocytochemistry. In some cells, xBest-2a was concentrated at and near the cell surface (Fig. 5, D-F). Fig. 5D (top) compares the distribution of xBest2a (green) with the distribution of wheat germ agglutinin (red). Wheat germ agglutinin (WGA) is seen as a sharp band at the cell surface and in intracellular compartments. xBest2a is also located at the cell surface and relatively little intracellular staining is observed. A plot of pixel intensity across the cell shows that WGA and xBest2a staining coincide very closely at the cell surface (Fig. 5, E and F). These results suggest that xBest2a can be expressed as a plasma membrane protein. In contrast, in other cells, immunofluorescent staining was concentrated in intracellular organelles that were identified as early endosomes by co-staining with an antibody to transferrin receptor (Fig. 5D, bottom). These cells usually had patches of fluorescence at or near the plasma membrane, as well, but the staining was often not uniform or light. We do not understand why the distribution appears to be different in different cells. Usually cells with pronounced endosomal staining were flat, large cells.

Ionic Currents of Heterologously Expressed xBest2a Currents
xBest-2a was transfected into cells along with a vector encoding for GFP expression. Cells expressing xBest-2a were identified by GFP fluorescence. Whole-cell patch clamp of xBest2a-expressing cells revealed that these cells had a Ca2+-dependent Cl- current that was not present in cells expressing GFP alone. Cells were patch-clamped in the whole-cell configuration with solutions having the same [Cl-] inside and outside. The major intracellular cation was Cs+, and the major extracellular cation was Na+. Under these conditions with intracellular free [Ca2+] buffered to <10 nM with 5 mM EGTA and 1 mM N,N'-(1,2-ethanediylbis-(oxy-2,1-phenylene))bis(N-(carboxymethyl))-tetrapotassium salt (BAPTA), the currents were small. In contrast, when free [Ca2+] was raised to 164 nM, currents were ~5-fold larger. Fig. 6A shows the averages for one of several experiments. For both xBest-2a- and xBest-2b-expressing cells, whole-cell currents with Ca2+ <10 nM were <400 pA. However, when intracellular free [Ca2+] was raised to 164 nM, currents ~1.5 nA were observed. In cells expressing GFP alone, the currents were <200 pA.



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  		FIG. 6.
Currents in xBest-2-expressing HEK-293 cells. xBest-2a or xBest-2b was transfected into HEK-293 cells as described under "Experimental Procedures." 1-2 days after transfection, the cells were whole-cell patch clamped. A, amplitude of steady-state currents in transfected cells. The steady-state currents during a 1-s voltage pulse to +100 mV was measured in cells either transfected with a GFP expression vector (pIRES2-EGFP), xBest-2a in pCMV-SPORT6 plus the GFP-expression vector at a ratio of 10:1, or the bi-cistonic construct pIRES2-EGFP encoding for xBest-2b and GFP. The cells were patched with a pipette solution containing either <10 nM free Ca2+ (open bars) or 164 nM free Ca2+ (hatched bars). B, steady-state currents in the presence of 20 µM tamoxifen to block swelling-activated currents. Transfections with GFP or xBest-2a plus GFP were performed, and cells were patched in the presence of 20 µM tamoxifen. Internal solution contained either <10 nM free Ca2+ or 400 nM free Ca2+ as indicated. C, current traces with <10 nM free Ca2+. Voltage protocol is shown above current traces. D, current traces with 400 nM free Ca2+. E, current-voltage relationships obtained with different free Ca2+ concentrations. Steady-state current at the beginning of the 350-ms pulse is plotted versus membrane potential. Squares, zero Ca2+; circles, 130 nM Ca2+; triangles, 180 nM Ca2+; inverted triangles, 250 nM Ca2+; diamonds, 400 nM Ca2+; side triangles, 4.5 µM Ca2+. F, dependence of the current on intracellular [Ca2+]. The amplitude of the average current at +100 mV was plotted versus [Ca2+]. n = 5-10 for each [Ca2+].

 
We were concerned that expression of xBest-2 might upregulate endogenous swelling-activated currents in the HEK-293 cells. One set of experiments was performed in cells bathed in 20 µM tamoxifen to block endogenous swelling-activated Cl- currents (28). Although the currents were smaller in the presence of tamoxifen, Ca2+ stimulated the currents ~6-fold (Fig. 6B).

Fig. 6, C-E shows current traces and current-voltage relationships from xBest-2a-expressing cells patched with different intracellular free [Ca2+]. With [Ca2+] < 10 nM, currents were very small (Fig. 6C). In contrast, with 400 nM free Ca2+, large time- and voltage-independent currents were observed (Fig. 6D). The currents were smaller at intermediate [Ca2+], but the waveforms of the currents were virtually the same. The current-voltage relationships for currents recorded with different intracellular free [Ca2+] are shown in Fig. 6E. The current-voltage relationships were linear for all [Ca2+] and reverse near 0 mV as expected for a Cl--selective current. A plot of current amplitudes versus [Ca2+] shows that the EC50 for Ca2+ is 210 nM (Fig. 6F). The currents recorded in cells expressing xBest-2b were very similar (Fig. 7). The calculated EC50 for xBest-2b was 228 nM.



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  		FIG. 7.
Currents in xBest-2b-expressing cells. The conditions were the same as in Fig. 8, except that the cells were transfected with pIRES-GFP vector encoding xBest-2b. A, current traces with <10 nM free Ca2+. B, current traces with 400 nM free Ca2+. C, current-voltage relationships of selected [Ca2+]. Squares, zero Ca2+; circles, 180 nM Ca2+; triangles, 250 nM Ca2+; inverted triangles, 4 µM Ca2+. D, dependence of the current on intracellular [Ca2+]. n = 3-10 for each [Ca2+].

 
Because the xBest currents exhibited a reversal potential near 0 mV, which was the same as the calculated ECl but was very different from the calculated equilibrium potentials of the major cations, Cs+ and Na+, we concluded that the Ca2+-activated current was a Cl- current. To confirm that the current was Cl--selective, extracellular Cl- was replaced with I-, Br-, or aspartate, and the shift in Erev was measured. Fig. 8 shows that the reversal potential shifted to the left for I- and Br- but shifted to the right for aspartate. These results suggest that the channel has a relative permeability ratio of I- > Br- > Cl- >> aspartate.



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  		FIG. 8.
Ionic selectivity of the current induced by xBest-2a. HEK-293 cells expressing xBest-2a were patched in whole-cell configuration. Extracellular Cl- was replaced with I-, Br-, or aspartate. A, current-voltage relationships. The current-voltage relationships were obtained as shown in Fig. 7 with I- (triangles), Br- (inverted triangles), Cl- (squares), or aspartate (circles). The reversal potentials shifted as expected for a Cl--selective channel with a permeability ratio, I- > Br- > Cl- >> Aspartate. B, the relative permeabilities were calculated from the Goldman-Hodgkin-Katz equation.

 
W93C and G229E Are Dominant Negative Mutations
To test whether the Cl- currents that appeared when we over-expressed xBest-2a were in fact mediated by this protein and were not caused by up-regulation of an endogenous current, we expressed two xBest-2a mutants. Both mutations (W93C and G299E) were selected, because these mutations in human Best-1 produce dominantly inherited Best vitelliform macular dystrophy (19). The W93C construct expressed alone produced negligible Ca2+-activated current (Fig. 9A, triangles). Furthermore, co-expression of W93C and wild type xBest-2a produced Ca2+-dependent currents that were 50% reduced in amplitude compared with those with wild type xBest-2a alone (Fig. 9A, circles). These data suggest that the W93C mutation is able to reduce the function of wild type protein. A more dramatic result was obtained with the G299E mutation. Expression of G299E alone produced no Ca2+-dependent current (Fig. 9B, circles). Expression of G299E with wild type suppressed the wild type current more than 90% (Fig. 9B, triangles). These data strongly suggest that xBest-2a is a subunit of a Ca2+-activated Cl- channel.



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  		FIG. 9.
W93C and G299E are dominant negative mutations. Open symbols, zero Ca2+ pipette solution. Closed symbols, high Ca2+ (4.5 µM) pipette solution. A, W93C mutation. HEK-293 cells were transfected with pIRES2-EGFP vector without bestrophin insert (inverted triangles), wild type xBest-2a (closed squares), W93C xBest-2a (triangles), and equal amounts of W93C and wild type xBest-2a together (circles). B, G299E mutation. HEK-293 cells were transfected with wild type xBest-2a (squares), G299E xBest-2a (circles), and equal amounts of wild type and G299E (triangles).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that Xenopus bestrophins produce Ca2+-dependent Cl- currents when they are expressed heterologously in HEK-293 cells. The bestrophin proteins are trafficked to the plasma membrane and produce currents that are anion-selective with a permeability sequence of I- > Br- > Cl- >> aspartate. The currents are activated by physiological concentrations of cytosolic Ca2+ (EC50 = ~210 nM). Two bestrophin mutations, W93C and G299E, produce non-functional channels that exert a dominant negative effect on the expression of wild type channels. On this basis, we conclude that Xenopus bestrophins are good candidates for a molecular component of Ca2+-dependent Cl- channels. In contrast, currents induced by the CLCA family are stimulated only by millimolar concentrations of Ca2+, and to date there have been no mutagenesis studies demonstrating an effect of point mutations on CLCA-induced currents.

The currents induced by heterologous expression of xBest-2a are similar to the native Ca2+-activated Cl- currents in Xenopus oocytes (10, 29). Both currents have EC50 values for Ca2+ in the range of 0.2 to 1 µM and exhibit similar anion permeability sequences (permeability ratios for I-:Br-:Cl-:aspartate are 4:2:1:0.7 for oocyte and 2.6:1.7:1:0.15 for xBest-2a). At saturating [Ca2+], both currents are time- and voltage-independent. However, there is a notable difference between the native current and the bestrophin current at [Ca2+] near the EC50. The native current has a voltage-dependent Ca2+ affinity such that at low [Ca2+], the current exhibits voltage-dependent gating behavior (29). This is not seen with the bestrophin current. However, Ca2+-activated Cl- currents reported in the literature differ significantly in their voltage-dependent characteristics. This suggests the possibility that, like other channels, bestrophin channel behavior might be modulated by other subunits.

Resemblance to GABA(A) Receptor—We identified bestrophin as a potential Ca2+-activated Cl- channel by its similarity to the pore domain of LGACs. The rationale was based on our previous studies (10, 12) that the native Ca2+-activated Cl- channel in Xenopus oocytes had very similar permeation properties to GABA(A) and glycine receptors in terms of anionic selectivity and estimated pore dimensions and orientation. The M2 domain of LGACs is related to what we believe is the 4th transmembrane domain of bestrophin. We call this region the RFP domain.

Although the RFP and M2 domains have important structural similarities, this does not imply that there is any evolutionary relationship between LGACs and bestrophins. There is no significant homology between these proteins in other regions, and according to our model, there are six transmembrane domains in bestrophins compared with four in LGACs. Furthermore, the homology between the RFP domain and the M2 domain are seen only when the sequences are compared in reverse orientation (the N-to C-terminal orientation of bestrophins is compared with the C to N orientation of the GABA(A) receptor in Fig. 1).

Our model of the topology of bestrophin differs from models suggested by others. Bakall et al. (30) and Sun et al. (20) suggested four transmembrane domains that correspond to transmembrane domains 1, 3, 5, and 6 in our model. The protein is proposed to have cytoplasmic N and C termini. White et al. (31) proposed five transmembrane domains that correspond to our transmembrane domains 2, 3, 5, and 6 plus a domain that we believe is in the cytoplasmic loop between transmembrane domains 4 and 5. They propose that the N terminus is cytoplasmic, and the C terminus is extracellular. The models by Bakall (30), Sun et al. (20), and White et al. (31) differ from our model in that their models place the RFP domain (our 4th transmembrane domain) in a cytoplasmic loop. However, the high degree of sequence conservation in the RFP domain, its hydrophobicity, and its similarity to the M2 domain of LGACs suggests that this region is involved in forming the pore. The importance of the RFP sequence is underlined by the fact that alanine mutations of the RFP sequence result in non-functional channels.2 Like the signature PAR sequence of LGACs, the RFP sequence is located at the cytoplasmic end of a transmembrane helix by several topology prediction algorithms.

In addition to the RFP-M2 similarity, another feature of bestrophins that attracted our attention to thinking that they might be Ca2+-activated Cl- channels was the highly conserved acidic domain immediately following the last transmembrane domain. Although this domain is only weakly related to known Ca2+ binding domains, its acidic nature suggested to us that it might be involved in Ca2+ binding.

Tissue Expression—By semi-quantitative PCR, it is very difficult to distinguish between xBest-2a and xBest-2b because of their high homology. We were unable to design primers for real-time PCR that were specific for the different orthologs. Message for xBest-2a and or xBest-2b is widely expressed. The tissues that expressed message most robustly were spleen, liver, RPE, neural retina, and lung. The A5925 antibody, which is specific for xBest-2a, shows that xBest-2a protein was expressed in spleen, gut, liver, and neural retina. Faint bands were observed in brain, heart, and lung. Thus, spleen, liver, and neural retina have high levels of both mRNA and protein, whereas brain and heart have much less. In contrast, in RPE the level of mRNA is high, but the level of protein is low. This could be explained if the mRNA in RPE was primarily xBest-2b. Because human bestrophin-1 is expressed almost exclusively in RPE, one might hypothesize that xBest-2b is playing the functional role in Xenopus RPE that bestrophin-1 plays in human.

Bestrophin Function—Best vitelliform macular dystrophy is an early onset form of macular degeneration that has been linked to over 80 different mutations in the human VMD2 gene that codes for bestrophin-1 (31). No other diseases have been reported to be linked to VMD2 mutations. These mutations tend to cluster, according to our model, in the 1st and 3rd transmembrane domains, near the acidic cluster following the last transmembrane domain, and in the second cytoplasmic loop. No disease-associated mutations have been reported in the C-terminal third of the protein or in the RFP domain. Perhaps mutations in the RFP domain produce a more serious defect that has not yet been identified. Many of the amino acids that when mutated cause Best disease are conserved in the Xenopus bestrophins. Because Best disease is an autosomal dominant disease, we tested the effect of two Best disease mutations on homologous residues in xBest-2a. Both the W93C and G299E mutations produce non-functional channels and reduce the currents produced by wild type xBest-2a. Not only do these results bolster our conclusion that bestrophins are channels, they also suggest that the channels form multimeric complexes and that the mutant channels have a dominant negative effect on channel function. How dysfunction of bestrophin channels may produce macular degeneration remains to be seen. The finding that human bestrophin-1 is located in retinal pigment epithelial cells has led to the suggestion that bestrophin may be the molecular counterpart of the basolateral Cl- channel in retinal pigment epithelial cells. Although we have tried to find a bestrophin in Xenopus that is more closely related to human bestrophin-1 than xBest-2a or xBest-2b, we have not succeeded. Our semi-quantitative PCR and Western blot results suggest that in Xenopus xBest-2b may play the same role as human bestrophin-1.

The high level of xBest-2a expression in neural retina suggests that this bestrophin may be a component of Ca2+-activated Cl- channels in photoreceptors or retinal neurons. In many species, both rods and cones have prominent Ca2+-activated Cl- currents (33-36). These currents have properties that are similar to the bestrophin currents we have described here. It has been suggested that the Ca2+-activated Cl- current in cones plays a role in modulating the voltage gain of the photoresponse and in mediating the effect of horizontal cell feedback (36). In rods, Ca2+ activated Cl- currents play a role in shaping and modulating the light response.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AY273825 [GenBank] and AY273826 [GenBank] .

* This work was supported in part by National Institutes of Health Grants GM-60448 and EY014852 [GenBank] (to H. C. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Cell Biology, Emory University School of Medicine, 535 Whitehead Biomedical Research Bldg., 615 Michael St., Atlanta, GA 30322. Tel.: 404-727-0444; Fax: 404-727-6256; E-mail: criss{at}cellbio.emory.edu.

1 The abbreviations used are: LGAC, ligand gated anion channels; RPE, retinal pigment epithelium; GST, glutathione S-transferase; xBest-2a, Xenopus bestrophin-2a (GenBankTM accession number AY273825 [GenBank] ); xBest-2b, Xenopus bestrophin-2b (GenBankTM accession number AY273826 [GenBank] ); GABA, {gamma}-aminobutyric acid; RT, reverse transcriptase; GFP, green fluorescent protein; EGFP, enhanced GFP; HEK, human embryonic kidney; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; WGA, wheat germ agglutinin. Back

2 Z. Qu, R. W. Wei, W. Mann, and H. C. Hartzell, unpublished information. Back



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 ABSTRACT
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 DISCUSSION
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The Bestrophin Mutation A243V, Linked to Adult-Onset Vitelliform Macular Dystrophy, Impairs Its Chloride Channel Function
Invest. Ophthalmol. Vis. Sci., November 1, 2006; 47(11): 4956 - 4961.
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L.-T. Chien, Z.-R. Zhang, and H. C. Hartzell
Single Cl- Channels Activated by Ca2+ in Drosophila S2 Cells Are Mediated By Bestrophins
J. Gen. Physiol., August 28, 2006; 128(3): 247 - 259.
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 Proc. Natl. Acad. Sci. USAHome page
S. Pifferi, G. Pascarella, A. Boccaccio, A. Mazzatenta, S. Gustincich, A. Menini, and S. Zucchelli
Bestrophin-2 is a candidate calcium-activated chloride channel involved in olfactory transduction
PNAS, August 22, 2006; 103(34): 12929 - 12934.
[Abstract] [Full Text] [PDF]

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T. Tsunenari, J. Nathans, and K.-W. Yau
Ca2+-activated Cl- Current from Human Bestrophin-4 in Excised Membrane Patches
J. Gen. Physiol., May 30, 2006; 127(6): 749 - 754.
[Abstract] [Full Text] [PDF]

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 J. Neurosci.Home page
Z. Qu, L.-T. Chien, Y. Cui, and H. C. Hartzell
The anion-selective pore of the bestrophins, a family of chloride channels associated with retinal degeneration.
J. Neurosci., May 17, 2006; 26(20): 5411 - 5419.
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L. Y. Marmorstein, J. Wu, P. McLaughlin, J. Yocom, M. O. Karl, R. Neussert, S. Wimmers, J. B. Stanton, R. G. Gregg, O. Strauss, et al.
The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1)
J. Gen. Physiol., April 24, 2006; 127(5): 577 - 589.
[Abstract] [Full Text] [PDF]

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 PhysiologyHome page
C. Hartzell, Z. Qu, I. Putzier, L. Artinian, L.-T. Chien, and Y. Cui
Looking Chloride Channels Straight in the Eye: Bestrophins, Lipofuscinosis, and Retinal Degeneration
Physiology, October 1, 2005; 20(5): 292 - 302.
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 Physiol. Rev.Home page
O. Strauss
The Retinal Pigment Epithelium in Visual Function
Physiol Rev, July 1, 2005; 85(3): 845 - 881.
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 Physiol. Rev.Home page
M. E. Loewen and G. W. Forsyth
Structure and Function of CLCA Proteins
Physiol Rev, July 1, 2005; 85(3): 1061 - 1092.
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 J. Physiol.Home page
R. Fischmeister and H. C. Hartzell
Volume sensitivity of the bestrophin family of chloride channels
J. Physiol., January 15, 2005; 562(2): 477 - 491.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Renal Physiol.Home page
A. Nissant, S. Lourdel, S. Baillet, M. Paulais, P. Marvao, J. Teulon, and M. Imbert-Teboul
Heterogeneous distribution of chloride channels along the distal convoluted tubule probed by single-cell RT-PCR and patch clamp
Am J Physiol Renal Physiol, December 1, 2004; 287(6): F1233 - F1243.
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A. D. Marmorstein, J. B. Stanton, J. Yocom, B. Bakall, M. T. Schiavone, C. Wadelius, L. Y. Marmorstein, and N. S. Peachey
A Model of Best Vitelliform Macular Dystrophy in Rats
Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3733 - 3739.
[Abstract] [Full Text] [PDF]

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S. R. Evans, W. B. Thoreson, and C. L. Beck
Molecular and Functional Analyses of Two New Calcium-activated Chloride Channel Family Members from Mouse Eye and Intestine
J. Biol. Chem., October 1, 2004; 279(40): 41792 - 41800.
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Z. Qu and C. Hartzell
Determinants of Anion Permeation in the Second Transmembrane Domain of the Mouse Bestrophin-2 Chloride Channel
J. Gen. Physiol., September 27, 2004; 124(4): 371 - 382.
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N. C. Robinson, P. Huang, M. A. Kaetzel, F. S. Lamb, and D. J. Nelson
Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current
J. Physiol., April 15, 2004; 556(2): 353 - 368.
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M. Pusch
Ca2+-activated Chloride Channels Go Molecular
J. Gen. Physiol., March 29, 2004; 123(4): 323 - 325.
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Z. Qu, R. Fischmeister, and C. Hartzell
Mouse Bestrophin-2 Is a Bona fide Cl- Channel: Identification of a Residue Important in Anion Binding and Conduction
J. Gen. Physiol., March 29, 2004; 123(4): 327 - 340.
[Abstract] [Full Text] [PDF]

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A. S. Piper and W. A. Large
Single cGMP-activated Ca2+-dependent Cl- channels in rat mesenteric artery smooth muscle cells
J. Physiol., March 1, 2004; 555(2): 397 - 408.
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V. V. Matchkov, C. Aalkjaer, and H. Nilsson
A Cyclic GMP-dependent Calcium-activated Chloride Current in Smooth-muscle Cells from Rat Mesenteric Resistance Arteries
J. Gen. Physiol., January 26, 2004; 123(2): 121 - 134.
[Abstract] [Full Text] [PDF]

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