Two Bestrophins Cloned from Xenopus laevis Oocytes Express Ca2+-activated Cl- Currents
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m308414200
ISSN1083-351X
AutoresZhiqiang Qu, Raymond W. Wei, W. P. Hei I Mann, H. Criss Hartzell,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoCa2+-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. 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. 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.Fuller C.M. Calcium-activated Chloride Channels. Academic Press, San Diego, CA2002Google Scholar). 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.Cunningham S.A. Awayda M.S. Bubien J.K. Ismailov I.I. Arrate M.P. Berdiev B.K. Benos D.J. Fuller C.M. J. Biol. Chem. 1995; 270: 31016-31026Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), 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.Gruber A.D. Elble R.C. Ji H.-L. Schreur K.D. Fuller C.M. Pauli B.U. Genomics. 1998; 54: 200-214Crossref PubMed Scopus (207) Google Scholar, 4.Gruber A.D. Schreur K.D. Ji H-L. Fuller C.M. Pauli B.U. Am. J. Physiol. 1999; 276: C1261-C1270Crossref PubMed Google Scholar, 5.Loewen M.E. Bekar L.K. Gabriel S.E. Walz W. Forsyth G.W. Biochem. Biophys. Res. Commun. 2002; 298: 531-536Crossref PubMed Scopus (30) Google Scholar, 6.Loewen M.E. Gabriel S.E. Forsyth G.W. Am. J. Physiol. Cell Physiol. 2002; 283: C412-C421Crossref PubMed Scopus (24) Google Scholar, 7.Romio L. Musante L. Cinti R. Seri M. Moran O. Zegarra-Moran O. Galietta L.J. Gene. 1999; 228: 181-188Crossref PubMed Scopus (50) Google Scholar). However, CLCA proteins have not been universally accepted as Ca2+-activated Cl- channels for several reasons (8.Jentsch T.J. Stein V. Weinreich F. Zdebik A.A. Physiol. Rev. 2002; 82: 503-568Crossref PubMed Scopus (1070) Google Scholar, 9.Papassotiriou J. Eggermont J. Droogmans G. Nilius B. Pflugers Arch. 2001; 442: 273-279Crossref PubMed Scopus (24) Google Scholar). 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) 1The abbreviations used are: LGACligand gated anion channelsRPEretinal pigment epitheliumGSTglutathione S-transferasexBest-2aXenopus bestrophin-2a (GenBank™ accession number AY273825)xBest-2bXenopus bestrophin-2b (GenBank™ accession number AY273826)GABAγ-aminobutyric acidRTreverse transcriptaseGFPgreen fluorescent proteinEGFPenhanced GFPHEKhuman embryonic kidneyGAPDHglyceraldehyde-3-phosphate dehydrogenasePIPES1,4-piperazinediethanesulfonic acidPBSphosphate-buffered salineWGAwheat germ agglutinin. of the GABA(A), GABA(C), and glycine receptor family (10.Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (152) Google Scholar, 11.Qu Z. Hartzell H.C. J. Biol. Chem. 2001; 276: 18423-18429Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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.Machaca K. Qu Z. Kuruma A. Hartzell H.C. McCarty N. Fuller, C.M. Calcium-activated Chloride Channels. Academic Press, San Diego, CA2002Google Scholar). ligand gated anion channels retinal pigment epithelium glutathione S-transferase Xenopus bestrophin-2a (GenBank™ accession number AY273825) Xenopus bestrophin-2b (GenBank™ accession number AY273826) γ-aminobutyric acid reverse transcriptase green fluorescent protein enhanced GFP human embryonic kidney glyceraldehyde-3-phosphate dehydrogenase 1,4-piperazinediethanesulfonic acid phosphate-buffered saline wheat germ agglutinin. 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.Lester H.A. Ann. Rev. Biophys. Biomol. Struct. 1992; 21: 267-292Crossref PubMed Scopus (129) Google Scholar, 14.Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar). 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.Langosch D. Laube B. Rundstrom N. Schmieden V. Bormann J. Betz H. EMBO J. 1994; 13: 4223-4228Crossref PubMed Scopus (151) Google Scholar) and intermediate rings (16.Xu M. Akabas M.H. J. Gen. Physiol. 1996; 107: 195-205Crossref PubMed Scopus (179) Google Scholar) 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.Keramidas A. Moorhouse A.J. French C.R. Schofield P.R. Barry P.H. Biophys. J. 2000; 78: 247-259Abstract Full Text Full Text PDF Scopus (102) Google Scholar, 18.Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (81) Google Scholar). Mutations affecting the proline and the positively charged intermediate ring can convert selectivity from anionic to cationic (17.Keramidas A. Moorhouse A.J. French C.R. Schofield P.R. Barry P.H. Biophys. J. 2000; 78: 247-259Abstract Full Text Full Text PDF Scopus (102) Google Scholar, 18.Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (81) Google Scholar). 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.Petrukhin K. Koisti M.J. Bakall B. Li W. Xie G. Marknell T. Sandgren O. Forsman K. Holmgren G. Andreasson S. Vujic M. Bergen A.A.B. McGarty-Dugan V. Figueroa D. Austin C.P. Metzker M.L. Caskey C.T. Wadelius C. Nat. Genet. 1998; 19: 241-247Crossref PubMed Scopus (584) Google Scholar). Bestrophin was initially thought to be involved in fatty acid transport (19.Petrukhin K. Koisti M.J. Bakall B. Li W. Xie G. Marknell T. Sandgren O. Forsman K. Holmgren G. Andreasson S. Vujic M. Bergen A.A.B. McGarty-Dugan V. Figueroa D. Austin C.P. Metzker M.L. Caskey C.T. Wadelius C. Nat. Genet. 1998; 19: 241-247Crossref PubMed Scopus (584) Google Scholar), but more recently (20.Sun H. Tsunenari T. Yau K.-W. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4008-4013Crossref PubMed Scopus (400) Google Scholar), 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.Francois J. De Rouck A. Fernandez-Sasso D. Arch. Ophthalmol. 1967; 77: 726-733Crossref PubMed Scopus (52) Google Scholar, 22.Deutman A.F. Arch. Ophthalmol. 1969; 81: 305-316Crossref PubMed Scopus (131) Google Scholar). 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.Gallemore R.P. Hughes B.A. Miller S.S. Marmor M.F. Wolfensberger T.J. The Retinal Pigment Epithelium. Oxford University Press, Oxford1998Google Scholar). Trans-RPE transport plays an important role in maintaining the fluid and ionic composition of the fluid surrounding the photoreceptors. Recently (24.Marmorstein A.D. Mormorstein L.Y. Rayborn M. Wang X. Hollyfield J.G. Petrukhin K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12758-12763Crossref PubMed Scopus (377) Google Scholar), 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. 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-β-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-free™ (Ambion). Semi-quantitative PCR—cDNA was synthesized from total RNA using ThermoScript™ 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 ThermoScript™ reverse transcriptase. The cDNA was amplified by using xBest-2a specific primers and SYBR Green JumpStart™ Taq ReadyMix™ (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 β-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.Tsien R.Y. Pozzan T. Methods Enzymol. 1989; 172: 230-262Crossref PubMed Scopus (399) Google Scholar. 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 × [Ca2+-EGTA]/[EGTA], where Kd is the Kd of EGTA (Kd = 1.0 × 10-7m 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 mmX and 11 mm Cl-, where X is the substitute anion. The permeability ratio was estimated using the Goldman-Hodgkin-Katz equation (26.Hille B. Ion Channels of Excitable Membranes, 2nd Ed. Sinauer Associates, Inc., Sunderland, MA1992Google Scholar), PX/PCl = [Cl-]i/([X]o exp(ΔErevF/RT)) - [Cl-]o/[X]o, where ΔErev 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 GenBank™ as accession number AY273825, and xBest-2b has been entered as AY273826. Other accession numbers are as follows: hBest1 (VMD2), NM_004183; hBest2 (VMD2L1), NM_017682; hBest3 (VMD2L2), NM_017682; hBest4 (VMD2L3), NM_152439. BLAST searching (27.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) 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) α1 subunit with four human bestrophins. The residues of the GABA(A) receptor are labeled in accordance with Keramidas (18.Keramidas A. Moorhouse A.J. Pierce K.D. Schofield P.R. Barry P.H. J. Gen. Physiol. 2002; 119: 393-410Crossref PubMed Scopus (81) Google Scholar) 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.Table IPhysical properties of the M2 domain of GABA receptor and xBest-2a RFP domainHuman GABA(A)R α1xBest2aIsoelectric point9.610.9Acidic (D,E)11Basic (K,R)24Polar (N,C,Q,S,T,Y)96Hydrophobic (A,I,L,F,W,V)1013 Open table in a new tab Because Ca2+-activated Cl- channels are expressed at high levels in Xenopus oocytes (12.Machaca K. Qu Z. Kuruma A. Hartzell H.C. McCarty N. Fuller, C.M. Calcium-activated Chloride Channels. Academic Press, San Diego, CA2002Google Scholar), we used the sequences obtained from mammalian bestrophins to find expressed sequence tags for Xenopus bestrophins. One Xenopus expressed sequence tag (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 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.Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar) (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.Fig. 3Properties 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.Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar). 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.2B, hydropathy analysis of xBest-2a by the Kyte-Doolittle algorithm (38.Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar) 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 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. 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 prote
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