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Two Bestrophins Cloned from Xenopus laevis Oocytes

Express Ca-activated Cl Currents.

Zhiqiang Qu, Raymond W. Wei, Wesley Mann, and H. Criss Hartzell†

Department of Cell Biology

Emory University School of Medicine

535 Whitehead Biomedical Research Building

615 Michael St.

Atlanta, GA 30322

† Corresponding author.

telephone: 404-727-0444

fax: 404-727-6256

email: criss@cellbio.emory.edu

Running title. Xenopus bestrophins are Ca-dependent Cl Channels

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 25, 2003 as Manuscript M308414200 by guest on D

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Summary

Ca-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 Ca-activated Cl channels which included members of the bestrophin

family. We cloned two bestrophins from Xenopus oocytes, which express high levels of Ca-

activated Cl channels. The Xenopus bestrophins were expressed in a variety of tissues. We

predict that bestrophin has 6 transmembrane domains with the conserved RFP domain

playing an integral part in ionic selectivity. When Xenopus bestrophins were heterologously

expressed in HEK-293 cells, large Ca-activated Cl currents were observed. The currents are

voltage-and time-independent, do not rectify, have a Kd for Ca 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 Ca in a physiological range.

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Introduction

Ca-activated Cl currents play a variety of important physiological roles which 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 Ca-activated Cl channels. Heterologous expression of hClCA1,

hCLCA2, mCLCA1, mCLCA2, bCLCA1, and pCLCA1 results in an increased Cl current that

is stimulated by intracellular Ca (3-7). However, CLCA proteins have not been universally

accepted as Ca-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

Ca concentrations and native expression of CLCAs does not correlate with Ca-activated Cl

currents.

Several years ago, we began an in silico approach to identify novel Cl channel genes.

Our previous studies on the endogenous Ca-activated Cl channels of Xenopus oocytes

suggested that these channels might have a pore architecture similar to ligand gated anion

channels (LGACs) of the GABA(A), GABA(C), and glycine receptor family (10;11).

LGACs and Ca-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 CFTR and ClC channels that seem to have the

wide end of their pores oriented cytoplasmically (12).

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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

3 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;19).

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

(20). Bestrophin was initially thought to be involved in fatty acid transport (20), but more

recently, it has been shown that human bestrophin-1 expressed in heterologous systems

induces a chloride current (21). The possibility that bestrophin is a Cl channel is particularly

exciting because the hallmark diagnostic feature of Best disease is an abnormal electro-

oculogram (22;23). 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) (24). Trans-RPE transport plays an important role in

maintaining the fluid and ionic composition of the fluid surrounding the photoreceptors.

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Recently, it has been demonstrated that human bestrophin-1 is localized to the basolateral

membrane of RPE (25), 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.

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Experimental Procedures

Cloning.

ESTs 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

Bgl II restriction site and the reverse primer contained a Sac II 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

(Quickchange, Stratagene). Specific mutations were introduced into primers. The template,

xBest-2a in pCMV-Sport6, was amplified with the primers and Pfu DNA polymerase by the

polymerase chain reaction. The methylated original templates were digested with Dpn 1 and

the PCR products were transformed into XL-1 Blue E. coli. Mutations were confirmed by

DNA sequencing.

Heterologous expession 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 Fugene 6 kit (Roche) or Ca-PO4 precipitation

(Clontech). Transfected cells were dissociated and replated one day after transfection and

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spread on glass coverslips. Fluorescent cells were used for patch clamp experiments within

three 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 6 of these 14 residues (VFQFPETVQDPPNN). Blast hits with an E value <10

included only xBest-2a. Peptides were synthesized by Research

Genetics (Invitrogen) using MAP resin technology to enhance antigenic response. The

peptides (with a cysteine added to the C-terminus) were conjugated to KLH, 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 was assayed by

ELISA 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 sub-cloning the

bestrophin C-terminal tail into pGEX-4T expression vectors (Amersham) 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 IPTG at final concentration

of 1mM for 3-4 hours at 37oC. Bacterial pellets were resuspended in 50mM Tris, pH 8.0,

40mM EDTA, 2.5% sucrose, 0.02% NaN3, 1mM PMSF, 2.5mg/ml lysozyme, and protease

inhibitor cocktail set II (Calbiochem, 5 µl/ 20 mg bacteria). The lysate was subjected to

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French press twice at 1000 psi. The lysate was centrifuged at 10,000 rpm for 15min, and

then passed over a glutathione Sepharose 4B columns (Amersham). The fusion proteins

were eluted from the columns with 10mM reduced Glutathione, 50mM 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 mono-phasic 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 DTT, 2U/µl RNaseOUT, and 0.75 U/µl

ThermoScriptTMRT. 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 (BioRad), and the protocol was: one cycle at 95oc for 2

min, 40 cycles at 95oc for 30 sec, 60oc for 30 sec, and 68oc for 30 sec. The primers for xBest-

2a were 5’ - TTG GCT GAA GGT GGG TGA ACA – 3’ and 5’ – GGG CGC GGG TCT

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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 (50mg tissue per ml) in the LSB buffer (60mM

NaCl, 25mM Na-PIPES pH 6.9, 1mM EDTA, 2mM NaN3, 0.3 mM β-mecaptoethanol, 0.1

mM PMSF and 100-fold diluted protease inhibitor cocktail 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 25mM 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, 200mM Glycine, 20% methanol.. The membranes

were blocked with 5% dry milk in PBS with 0.1% Tween 20 (PBS-T) overnight at 4oC or 1

hr at room temperature. The blot was incubated with primary antibody (1/1000 dilution) and

horseradish peroxidase conjugated goat anti-rabbit IgG (1/7000) (Jackson Immuno Research)

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 hr. The cells were washed 3 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-

X100, 3% bovine serum albumin and 10% normal goat serum for 30 min. The cells were

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then incubated at 4oC overnight with 180 ng/ml affinity-purified A5925 in PBS containing

2% BSA 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 pipets had resistances of 3 - 5 MΩ filled with the standard intracellular solution (see

below). The bath was grounded via a 3M 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-msec duration pulses from

-100 mV to +100 mV. To measure the instantaneous current-voltage relationship, a pre-pulse

was first applied to +100mV for 200ms and then 500 msec duration voltage steps were

applied between -100 mV 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 (mM): 146 CsCl, 2 MgCl2, 5 (Ca)-EGTA, 8

HEPES, 10 sucrose, pH 7.3. The “zero” free Ca2+ pipette solution contained 5 mM EGTA

without added Ca2+ while high free Ca2+ pipette solution contained 5mM Ca-EGTA made by

the pH-metric method described by (26). Working solutions having different free Ca2+ were

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prepared by mixing the zero-Ca2+ solution with the high Ca2+ solution in various ratios. The

free [Ca2+] was calculated from the equation

[Ca2+] = K d x [Ca-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 (Perkin Elmer, Norwalk

CT). In some experiments, pipette solution contained (mM) 148 CsCl, 2 MgCl2, 0.5 CaCl2, 5

EGTA, 8 HEPES, pH 7.3 with free [Ca] = 164 nM.

The standard extracellular solution contained (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, Massachusetts, USA). 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 (27).

Px / PCl = [Cl]i / ([X]o exp(∆ErevF/RT))-[Cl] o / [X] o

where ∆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.

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Accession numbers.

XBest-2a has been enetered in Genbank as accession AY273825 and XBest-2b has

been entered as AY273826. hBest1 (VMD2): NM_004183, hBest2 (VMD2L1):

NM_017682, hBest3 (VMD2L2): NM_017682, hBest4 (VMD2L3): NM_152439.

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Results

In silico cloning of Ca-activated Cl Channels.

Blast searching (28) of protein databases 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) α1 subunit with four human bestrophins. The residues of the

GABA(A) receptor are labeled in accordance with Keramidas (29) 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’ R of the 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 hGABA(A)R and the RFP

domain of xBest-2a are quite similar (Table 1). 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 C. elegans to human. This strongly suggests that the RFP

sequence is crucial to channel function. We propose it is involved in ion selectivity.

Because Ca-activated Cl channels are expressed at high levels in Xenopus oocytes

(12), we used the sequences obtained from mammalian bestrophins to find ESTs for Xenopus

bestrophins. One Xenopus EST (BE669309, 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 3 overlapping sequencing runs were performed. The PCR products

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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 was determined by the neighbor joining method of Saitou and Nei (1987) (Fig. 3A).

The bestrophins from Zebrafish, Fugu, Xenopus, mouse, rat, and human fall into 4 groups

with the both Xenopus bestrophins being in group 2. The two Xenopus bestrophins are 89%

identical. Because Xenopus 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.

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 PKA, casein kinase, and PKC

phosphorylation consensus (PROSITE) sequences, but only three were conserved in the

human proteins (Fig. 2). T164 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. T6 and T216 are predicted PKC phosphorylation sites found in 4 and 3,

respectively, of the bestrophins shown here. The other phosphorylation sites were not

conserved across species.

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Kyte-Doolittle hydropathy analysis of xBest-2a with a window of 17 found 6

hydrophobic domains (Fig. 3B). The SOSUI algorithm predicted 6 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, 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.

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 54kD band in

neural retina, liver, gut, and spleen (Fig. 5B). A faint 54kD band was also observed in brain,

heart, lung, and oocyte. No band was observed in RPE. The 54kD band was absent when the

antibody was pre-absorbed with the GST-xBest-2a fusion protein (Fig. 5C). The mass of

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XBest-2a predicted from the cDNA sequence is 59kD. We presume that the 54kD band

corresponds to the predicted 59kD protein.

Expression in HEK cells. xBest2a was expressed in HEK293 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. 5D-F). Figure 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. 5E-F). These results

suggest that xBest2a can be expressed as a plasma membrane protein. In contrast, in other

cells, immunoflourescent staining was concentrated in intracellular organelles that were

identified as early endosomes by co-staining with an antibody to transferrin receptor (Figure

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 Ca-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

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cation was Cs and the major extracellular cation was Na. Under these conditions with

intracellular free [Ca] buffered to <10 nM with 5 mM EGTA and 1 mM BAPTA, the currents

were small. In contrast, when free [Ca] 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 Ca <10 nM were <400 pA. However, when

intracellular free [Ca] was raised to 164 nM, currents ~1.5 nA were observed. In cells

expressing GFP alone, the currents were <200 pA.

We were concerned that expression of xBest-2 might up-regulate 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 (30).

Although the currents were smaller in the presence of tamoxifen, Ca stimulated the currents

~6-fold (Fig. 6B).

Figure 6 C-E shows current traces and current-voltage relationships from xBest-2a

expressing cells patched with different intracellular free [Ca]. With [Ca] < 10 nM, currents

were very small (Fig. 6C). In contrast, with 400 nM free Ca, large time- and voltage-

independent currents were observed (Fig. 6D). The currents were smaller at intermediate

[Ca], but the waveforms of the currents were virtually the same. The current-voltage

relationships for currents recorded with different intracellular free [Ca] are shown in Fig. 6E.

The current-voltage relationships were linear for all [Ca] and reverse near 0 mV as expected

for a Cl-selective current. A plot of current amplitudes vs. [Ca] shows that the EC50 for Ca 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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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 Ca-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.

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 (20). The W93C construct expressed alone produced

negligible Ca-activated current (Fig. 9A, triangles). Furthermore, co-expression of W93C and

wild-type XBest-2a produced Ca-dependent currents that were 50% reduced in amplitude

compared to 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 Ca-

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 Ca-activated Cl channel.

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Discussion

We have shown that Xenopus bestrophins produce Ca-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 Ca (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 Ca-dependent Cl channels. In contrast,

currents induced by the CLCA family are stimulated only by millimolar concentrations of Ca,

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

Ca-activated Cl currents in Xenopus oocytes (10;31). Both currents have EC50’s for Ca 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 [Ca], both currents are time-and voltage independent. However, there is a notable

difference between the native current and the bestrophin current at [Ca] near the EC50. The

native current has a voltage-dependent Ca affinity such that at low [Ca], the current exhibits

voltage-dependent gating behavior (31). This is not seen with the bestrophin current.

However, Ca-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.

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Resemblance to GABA(A) receptor.

We identified bestrophin as a potential Ca-activated Cl channel by its similarity to the

pore domain of LGACs. The rationale was based on our previous studies that the native Ca-

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 (10;12). 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 6 transmembrane domains in bestrophins compared to 4 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 to 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. (32) and Sun et al. (21) suggested 4 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. (33) proposed 5 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 (34), Sun

et al. (21), and White et al. (33) 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

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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 (unpublished). 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 Ca-activated Cl channels was the highly

conserved acidic domain immediately following the last transmembrane domain. Although

this domain is only weakly related to known Ca binding domains, its acidic nature suggested

to us that it might be involved in Ca 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

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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 (33;35). 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 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 lead 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

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xBest-2a or xBest-2b, we have not succeeded. Our semi-quanititative 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 Ca-activated Cl channels in photoreceptors or retinal neurons. In many species,

both rods and cones have prominent Ca-activated Cl currents (36-39). These currents have

properties that are similar to the bestrophin currents we have described here. It has been

suggested that the Ca-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 (40). In

rods, Ca activated Cl currents play a role in shaping and modulating the light response.

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Acknowledgements

Supported by NIH grant GM-60448 to HCH.

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Footnotes

Abbreviations: LGAC: ligand gated anion channels, CFTR:, cystic fibrosis

transmembrane conductance regulator, RPE: retinal pigment epithelium, PCR: polymerase

chain reaction, GST: glutathione S-transferase, XBest-2a: Xenpous bestrophin-2a

(GENBANK AY273825), XBest-2b: Xenbopus bestrophin-2b (GENBANK AY273826).

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Figure Legends

Figure 1. Comparison of human GABA(A) receptor α1 subunit M2 domain with the

bestrophin RFP domain. The M2 domain of the human GABA(A)α1 subunit is shown from

residue 301 – 277. The intermediate positively charged ring at R282 is labeled 0’. (17). The

outer positively charged ring is R301 at position 19’ and the negatively charged inner ring is

E277 at position -5’. The bestrophins are shown from residue 130 – 155. Identical residues

are highlighted in red, similar residues in orange, and weakly similar residues in blue.

Figure 2. Alignment of human and Xenopus bestrophins. Identical residues are shaded.

Transmembrane domains predicted by the SOSUI algorithm (41) are indicated by horizontal

lines. Consensus sequences for phosphorylation are highlighted in green for PKA, red for

PKC, and blue for casein kinase II. The signature RFP residues are cyan. The last 44 residues

of hBest-1 are not shown. Genbank accession numbers are xBest-2a: AY273825, xBest-2b:

AY2273826, hBest-1: NM_004183, hBest-2: NM_017682, hBest-3: NM_017682, hBest-4:

NM_152439.

Figure 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 (1987). Accession numbers

as in Figure 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 lab (unpublished).

(B). Hydropathy analysis of xBest-2a by the Kyte-Doolittle algorithm (42) with a window of

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17 residues shows 6 distinct hydrophobic domains. This finding, coupled with the results of

the SOSUI transmembrane prediction shown in Fig. 2, suggests 6 transmembrane domains.

(C). Proposed topology of xBest-2a. Transmembrane domains are shaded in 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.

Figure 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

to the amount of GAPDH message as described in Methods.

Figure 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), visualized with peroxidase-conjugated goat anti-rabbit

secondary antibody by chemi-luminescence. (B). Various Xenopus tissues were

homogenized, dissolved in SDS buffer (methods), 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 2 hours 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

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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 wheat-germ agglutinin (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.

Figure 6. Currents in xBest-2 expressing HEK-293 cells. xBest-2a or xBest-2b was

transfected into HEK-293 cells as described in methods. 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-sec voltage pulse to +100 mV was measured in cells

either transfected with (a) a GFP expression vector (pIRES2-EGFP), (b) xBest-2a in pCMV-

SPORT6 plus the GFP-expression vector at a ratio of 10 : 1, or (c) the bi-cistonic construct

pIRES2-EGFP encoding for xBest-2b and GFP. The cells were patched with a pipet solution

containing either <10 nM free Ca (open bars), or 164 nM free Ca (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

Ca or 400 nM free Ca as indicated. (C) Current traces with <10 nM free Ca. Voltage

protrocol is shown above current traces. (D). Current traces with 400 nM free Ca. (E)

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Current-voltage relationships obtained with different free Ca concentrations. Steady state

current at the beginning of the 350 msec pulse is plotted vs. membrane potential. squares:

zero Ca, circles: 130 nM Ca, up triangles: 180 nM Ca, down triangles: 250 nM Ca,

diamonds: 400 nM Ca, side triangles: 4.5 µM Ca. (F) Dependence of the current on

intracellular [Ca]. The amplitude of the average current at +100 mV was plotted vs. [Ca]. n=

5-10 for each [Ca].

Figure 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 Ca. (B) Current traces with 400 nM free Ca. (C) Current-voltage

relationships of selected [Ca]. Square: zero Ca, circle: 180 nM Ca, up triangle: 250 nM Ca,

down triangle: 4 µM Ca. (D). Dependence of the current on intracellular [Ca]. n= 3-10 for

each [Ca].

Figure 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 either

I, Br, or aspartate. (A) Current-voltage relationships. The current-voltage relationships were

obtained as shown in Fig. 7 with I (up triangles), Br (down triangles), Cl (squares), or

asparate (circles). The reversal potentials shifted as expected for a Cl-selective channel with

pI>pBr>pCl>>pAspartate. B. The relative permeabilities were calculated from the GHK

equation:

Px / PCl = [Cl]i / ([X]o exp (∆ErevF/RT))-[Cl] o / [X] o.

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Figure 9. W93C and G299E are dominant negative mutations. Open symbols: zero Ca pipet

solution. Solid symbols: High Ca (4.5 µM) pipet solution. (A) W93C mutation. HEK-293

cells were transfected with pIRES2-EGFP vector without bestrophin insert (down triangles),

wild type xBest-2a (solid squares), W93C xBest-2a (up 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).

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Table 1. Physical Properties of the M2 domain of GABA receptor and xBest-2a.

hGABA(A)R α1 xBest2a

Isoelectric point 9.6 10.9

acidic (D,E) 1 1

basic (K,R) 2 4

polar (N,C,Q,S,T,Y) 9 6

hydrophobic (A,I,L,F,W,V) 10 13

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Fig.1 19’ 0’-2’ -5’ hGABAA (301) R-ASISLTTMTLVTTVGFVTRAPVSE hBest1 (130) RYANLGNVLILRSVSTAVYKRFPSAQ hBest2 (130) RYAGLSAVLILRSVSTAVFKRFPTID hBest3 (130) RYANLASVLVLRSVSTRVLKRFPTME hBest4 (130) RYVNLTSLLIFRSVSTAVYKRFPTMD xbest2 (130) RYCSLSGLLILRSVSTAVFKRFPTID + + P - OUTER INTERM INNER

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Fig.2 hBest-1 1 MTITYTSQVA NARLGSFSRL LLCWRGSIYK LLYGEFLIFL LCYYIIRFIY RLALTEEQQL MFEKLTLYCD hBest-2 1 MTVTYTARVA NARFGGFSQL LLLWRGSIYK LLWRELLCFL GFYMALSAAY RFVLTEGQKR YFEKLVIYCD hBest-3 1 MTVSYTLKVA EARFGGFSGL LLRWRGSIYK LLYKEFLLFG ALYAVLSITY RLLLTQEQRY VYAQVARYCN hBest-4 1 MTVTYSSKVA NATFFGFHRL LLKWRGSIYK LLYREFIVFA VLYTAISLVY RLLLTGVQKR YFEKLSIYCD XBest-2b 1 MTVTYTARVA NARFGGFYKL LLLWRGSIYK LLYKELLAFF IMYLALSITY RFFLNEEQRL YFEKVAIYCN XBest-2a 1 MTVTYTARVA NARFGGFYKL LLLWRGSIYK LLYKEFLAFL IMYLGLSIIY RFLLNDEQRL YFEKVAIYCN hBest-1 71 SYIQLIPISF VLGFYVTLVV TRWWNQYENL PWPDRLMSLV SGFVEGKDEQ GRLLRRTLIR YANLGNVLIL hBest-2 71 QYASLIPVSF VLGFYVTLVV NRWWSQYLCM PLPDALMCVV AGTVHGRDDR GRLYRRTLMR YAGLSAVLIL hBest-3 71 RSADLIPLSF VLGFYVTLVV NRWWSQYTSI PLPDQLMCVI SASVHGVDQR GRLLRRTLIR YANLASVLVL hBest-4 71 RYAEQIPVTF VLGFYVTLVV NRWWNQFVNL PWPDRLMFLI SSSVHGSDEH GRLLRRTLMR YVNLTSLLIF XBest-2b 71 NYANLIPVSF VLGFYVTLVV SRWWNQYLSI PFPDRVMCAI SGTVHGSDEK GRLYRRTLMR YCSLSGLLIL XBest-2a 71 NYANLIPVSF VLGFYVTLVV NRWWNQYLCM PFPDRVMCAV SGTVHGSDET GRLYRRTLIR YCSLSGLLIL hBest-1 141 RSVSTAVYKR FPSAQHLVQA GFMTPAEHKQ LEKLSLPHNM FWVPWVWFAN LSMKAWLGGR IRDPILLQSL hBest-2 141 RSVSTAVFKR FPTIDHVVEA GFMTREERKK FENLNSSYNK YWVPCVWFSN LAAQARREGR IRDNSALKLL hBest-3 141 RSVSTRVLKR FPTMEHVVDA GFMSQEERKK FESLKSDFNK YWVPCVWFTN LAAQARRDGR IRDDIALCLL hBest-4 141 RSVSTAVYKR FPTMDHVVEA GFMTTDERKL FNHLKSPHLK YWVPFIWFGN LATKARNEGR IRDSVDLQSL XBest-2b 141 RSVSTAVFKR FPTIDHVVEA GFMTRLERKK FENLQSSYNK YWVPCVWFCN LAAQGRSEGR IRDDHSFKML XBest-2a 141 RSVSTAVFKR FPTIDHVVEA GFMTRLERKK FENLQSSYNK YWVPCVWFCN LAAQARSEGR IRDDHSFKML hBest-1 211 LNEMNTLRTQ CGHLYAYDWI SIPLVYTQVV TVAVYSFFLT CLVGRQFLNP AKAYPG---- ---------- hBest-2 211 LEELNVFRGK CGMLFHYDWI SVPLVYTQVV TIALYSYFLA CLIGRQFLDP AQGYKD---- ---------- hBest-3 211 LEELNKYRAK CSMLFHYDWI SIPLVYTQVV TIAVYSFFAL SLVGRQFVEP EAGAAKPQKL LKPGQEPAPA hBest-4 211 MTEMNRYRSW CSLLFGYDWV GIPLVYTQVV TLAVYTFFFA CLIGRQFLDP TKGYAG---- ---------- XBest-2b 211 MEELNTFRGN CSMLFHHDWI SVPLVYTQVV TIAVYSFFLT CLIGRQFLDP LRGYPG---- ---------- XBest-2a 211 MEELNTFRGN CGMLFHYDWI SIPLVYTQVV TIAVYSFFLT CLIGRQFLDP SRGYPG---- ---------- hBest-1 267 -HELDLVVPV FTFLQFFFYV GWLKVAEQLI NPFGEDDDDF ETNWIVDRNL QVSLLAVDEM HQDLPRMEPD hBest-2 267 -HDLDLCVPI FTLLQFFFYA GWLKVAEQLI NPFGEDDDDF ETNFLIDRNF QVSMLAVDEM YDDLAVLEKD hBest-3 282 LGDPDMYVPL TTLLQFFFYA GWLKVAEQII NPFGEDDDDF ETNQLIDRNL QVSLLSVDEM YQNLPPAEKD hBest-4 267 -HDLDLYIPI FTLLQFFFYA GWLKVAEQLI NPFGEDDDDF ETNWCIDRNL QVSLLAVDEM HMSLPKMKKD XBest-2b 267 -HELDLYIPV FTLLQFFFYA GWLKVGEQLI NPFGEDDDDF ETNFLIDRNF QVSMLAVDEM YSDVPPMEKD XBest-2a 267 -HELDLYIPV FTLLQFFFYA GWLKVGEQLI NPFGEDDDDF ETNFLIDRNF QVSMLAVDEM YSDVPPMEKD hBest-1 336 MYWNKPEPQP PYTAASAQFR -RASFMGSTF NISLNKEEME FQPNQ-E--D EEDAHAGIIG RFLGLQSHDH hBest-2 336 LYWDAAEARA PYTAATVFQL RQPSFQGSTF DITLAKEDMQ FQRLDGLDGP MGEAPGDFLQ RLLPAGAGMV hBest-3 351 QYWDEDQPQP PYTVATAAES LRPSFLGSTF NLRMSDDPEQ SLQVEAS--- -PGSGRPAPA AQTPLLGRFL hBest-4 336 IYWDDSAARP PYTLAAADYC -IPSFLGSTV QMGKQMP--- ---------K -NEWKMEDIK IPLPQPQFQC XBest-2b 336 RYWNHSDPRP PYTAATLFQK HLPSFQGSTF NMAIPKEDMQ FQPLS----D IEEMNEDTLT HTLPLFSRFP XBest-2a 336 RYWNHSDPRP PYTAATLFQK HLPSFQGSTF NMAIPKEDMQ FQPLS----D IEEMNEDTLM HPLPLLSRFL hBest-1 402 HPPRANSRTK LLWPKRESLL HEGLPKNHKA AKQNVRGQED NKAWKLKAVD AFKSAPLYQR PGYYSAPQTP hBest-2 406 AGGPLGRRLS FLLRKNSCVS EASTGASCSC AVVPEG---- ------AAPE CSCGDPLLDP --GLPEPEAP hBest-3 417 GVGAPSPAIS LRNFGRVRGT PRPPHLLRFR AEEGGD---- ------PEAA ARIEEESAES GDEALEP--- hBest-4 392 AKSDPGG--- ---------- ---------- ---------- ---------- ---------- ---------- XBest-2b 402 PGIGPSPLSS SAGLSTLFAA PGSRLTLLSH STSSFS---- ------SSSV FQFPETVQDP PNNLVDPTAP XBest-2a 402 PGVGPSPLSS SAALAAQFAA PENNFTLLRR STSAFS---- ------SSSE FQSQEPIQDP PYNLVDTKTP hBest-1 472 LSPTPMFFPL EPSAPSKLHS VTGIDTKDKS LKTVSSGAKK SFELLSESDG ALMEHPEVSQ VRRKTVEFNL +44 hBest-2 462 PPAGPEPLTL IPGPVEPFSI VTMPGPRGPA PPWLPSPIGE EEENLA---- ---------- ---------- hBest-3 ---------- ---------- ---------- ---------- ---------- ---------- ---------- hBest-4 ---------- ---------- ---------- ---------- ---------- ---------- ---------- XBest-2b 462 GLNVQEWPLE KLFSIGSQNS LFHPPKTMDG CGNVQSVMPE EAEDAASLVA T--------- ---------- XBest-2a 462 ELNVQECQIE KQFSVGSQVS LFLPPKTMDI DGNIQSVMPE EEEDTASLVA T--------- ---------- PKA PKC casein

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XBest-1 XBest-2

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177

11481645037

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0 1 0 0 2 0 0 3 0 0- 3- 2- 1012

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-100 -50 50 100

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-3

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wt, 0Ca, n=5 wt, High Ca, n=8 G299E,High Ca, n=7 wt + G299E, High Ca, n=7

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EGFP, High Ca, n=5 wt, High Ca, n=6 W93C, High Ca, n=7 wt + W93C, High Ca, n=6 wt + W93C, 0 Ca, n=6

Vm (mV)

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B

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Zhiqiang Qu, Raymond W Wei, Wesley Mann and H. Criss HartzellTwo bestrophins cloned from Xenopus laevis Oocytes express Ca-activated Cl currents

published online August 25, 2003J. Biol. Chem.

10.1074/jbc.M308414200Access the most updated version of this article at doi:

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