Two Bestrophins Cloned from Xenopus laevis Oocytes Express Ca2+-activated Cl- Currents

Abstract

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- 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 in Best is thought to an in Cl- conductance the of the retinal pigment epithelium T.J. The Press, Scholar). transport an important role in the and ionic of the the A.D. M. K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), it has been that human bestrophin-1 is to the of it a for the Cl- channel of In this we the and expression of two bestrophins from Xenopus proteins have high to human bestrophins and Cl- currents expressed in heterologous sequence for Xenopus bestrophins were identified by conserved domains of bestrophins. were and Xenopus bestrophins were obtained by from Xenopus The were and in both and reverse was at three was by The a and the reverse a for the of mutations were a mutations were The in was with the by the The were with and the were Mutations were by Heterologous of in in was with at a ratio of was in was with or were and and on were used for were a of The acid sequence was which to of in six of these residues hits with an included were by to The a to the were to with an of and two The a and were by and with proteins of bestrophins. The were proteins of several bestrophins were by the bestrophin expression with The used for and were residues The protein of by the of the The proteins were in and by at a of for at were in and The was to French at The was at for and a The proteins were from the with and from Xenopus and were a of and was a The was two with and the was by with was by and was from reverse transcriptase with The of and reverse The was by and The of was were with which the for The of the was The of the is by the ratio of the of the in the The used for was and the was as at for at for for and for The for were and The for were and and were and were and The were in the and was to a of was by the a was by the and with in were on in with of protein The proteins were to in The were blocked with in with at or at The was with and in with was by enhanced were in in for The were three with and blocked with bovine and for The were at with in bovine The were with and with The were a were the of the were of by a and of with the intracellular The was a to a were by of the at a of the the were from a potential of with from to the a was first to for and were and were by an by a were at the The Ca2+ Ca2+ whereas high Ca2+ by the by in T. PubMed Scopus Google Ca2+ were by the with the high Ca2+ in The was from the Kd Kd is the Kd of at ionic The Ca2+ were in by an In some the with The extracellular the When Cl- was with was with is the was for both and extracellular in were by of of the and we used permeability of the channels was by the in the from Cl- to with and is the The permeability ratio was the B. of Scholar), is the the potential with the anion and that with and and have their has been in as accession number and has been as accession are as searching W. W. D.J. J. Biol. PubMed Scopus Google Scholar) of protein with degenerate M2 domains of LGACs a number of proteins of the bestrophin family. an of the M2 domain of the human with human bestrophins. The residues of the receptor are in with 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 of the intermediate and extracellular rings at position and The the bestrophins and the receptor in this is However, the residues for receptor ion selectivity are the extracellular the of the intermediate the negative of the and the -2′ proline at the pore The properties of the M2 domain of the human and the RFP domain of are similar the RFP sequence to of the bestrophins that to the intermediate ring and proline of the receptor is members of the bestrophin family for which sequences are from to This that the RFP sequence is to channel We it is involved in ion properties of the M2 domain of receptor and RFP in a 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 bestrophins to expressed sequence for Xenopus bestrophins. Xenopus expressed sequence was obtained and and to be a The sequence was by obtained by of from Xenopus three were The from in sequence from were to a the sequences of the two Xenopus bestrophins to human bestrophins. bestrophins for which sequence is were by the of and N. M. Biol. Google Scholar) The bestrophins from and human with the both Xenopus bestrophins in The two Xenopus bestrophins are is and and are more to of the human bestrophins are to we that the Xenopus bestrophins are of of bestrophins. The Xenopus human and bestrophins were the of and N. M. Biol. Google Scholar). are as in that sequences were obtained from the is is and is The bestrophins were cloned in of by the J. J. Biol. PubMed Scopus Google Scholar) with a of residues six This with the results of the transmembrane shown in six transmembrane proposed of domains are charged residues are negatively charged residues are and proline is the of the RFP sequence at the end of transmembrane bestrophins are conserved in the first residues but differ in the of the is to in the first but in the is to in the first but from residue to the The Xenopus bestrophins protein and protein but three were conserved in the human proteins is a that is conserved in six bestrophins shown and is conserved in a of the bestrophins we have and are protein in and of the bestrophins shown The were not conserved of with a of six domains The six transmembrane domains as the sequence in The sequence is at the end of the transmembrane domain and be to be at the of the for bestrophin was used to the expression of The that were were to sequences in both and the the of both is expressed at high levels in and and to a in and was in and we were to and from from we were to the of by were to sequences of the Xenopus bestrophins. was residues of This proteins of the of but not with Xenopus that the a in and was in and was in The was the was with the protein The of from the sequence is We that the to the in was expressed in by We two of by In some cells, was at and the cell the of with the of wheat germ germ is as a at the cell and in intracellular is at the cell and intracellular is observed. of the cell that and very at the cell and results that can be expressed as a In in cells, was in intracellular that were identified as early by with an to receptor of at or the as but the was not or We do not the to be in with were large was with a for GFP were identified by GFP of that these a Cl- current that was not in GFP were in the with the and The major intracellular was and the major extracellular was these with intracellular to with and the currents were In was to nm, currents were the for of several both and cells, currents with Ca2+ were However, intracellular was to nm, currents were observed. In GFP the currents were We were that expression of might endogenous currents in the of was in in to block endogenous Cl- currents B. Eggermont J. T. G. V. Droogmans G. Biophys. Biol. PubMed Scopus Google Scholar). Although the currents were in the of Ca2+ stimulated the currents current and from with intracellular nm, currents were very In with large and currents were The currents were at intermediate but the of the currents were the The for currents with intracellular are shown in The were for and reverse as for a current. of current that the for Ca2+ is The currents in were very similar The for was the currents a potential which was the as the but was very from the of the major and we that the Ca2+-activated current was a Cl- current. that the current was extracellular Cl- was with or and the in was that the potential to the for I- and Br- but to the for aspartate. results that the channel has a permeability ratio of I- > Br- > Cl- >> aspartate. the Cl- currents that we were in by this protein and were not by of an endogenous we expressed two mutations and were because these mutations in human produce inherited 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). The W93C expressed Ca2+-activated current Furthermore, of W93C and wild type currents that were in with with wild type that the W93C is to the of wild type more was obtained with the G299E of G299E current of G299E with wild type the wild type current more that is a of a Ca2+-activated Cl- We have shown that Xenopus bestrophins produce Cl- currents they are expressed heterologously in The bestrophin proteins are to the and produce currents that are with a permeability sequence of I- > Br- > Cl- >> aspartate. The currents are activated by physiological of Ca2+ ∼210 bestrophin W93C and produce non-functional channels that exert a dominant negative effect on the expression of wild type channels. this we conclude that Xenopus bestrophins are for a molecular of Cl- channels. In currents by the CLCA family are stimulated by of and to have been studies an effect of point mutations on CLCA-induced currents. The currents by heterologous expression of are similar to the native Ca2+-activated Cl- currents in Xenopus oocytes (10.Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (152) Google Scholar, A. Hartzell H.C. J. Gen. Physiol. 2000; PubMed Scopus Google Scholar). currents have for Ca2+ in the of to and exhibit similar anion permeability sequences for are for and for both currents are and However, is a the native current and the bestrophin current at the The native current has a Ca2+ that at the current A. Hartzell H.C. J. Gen. Physiol. 2000; PubMed Scopus Google Scholar). This is not with the bestrophin current. However, Ca2+-activated Cl- currents in the differ in their This the possibility channels, bestrophin channel might be by to identified bestrophin as a potential Ca2+-activated Cl- channel by to the pore domain of The was on previous studies (10.Qu Z. Hartzell H.C. J. Gen. Physiol. 2000; 116: 825-844Crossref PubMed Scopus (152) Google Scholar, K. Qu Z. Kuruma A. Hartzell H.C. McCarty N. Fuller, C.M. Calcium-activated Chloride Channels. Academic Press, San Diego, CA2002Google Scholar) that the native Ca2+-activated Cl- channel in Xenopus oocytes very similar properties to and glycine in of anionic selectivity and pore and The M2 domain of LGACs is to we is the transmembrane domain of We this the RFP Although the RFP and M2 domains have important this does not that is LGACs and bestrophins. is these proteins in and to are six transmembrane domains in bestrophins with in Furthermore, the the RFP domain and the M2 domain are the sequences are in reverse of bestrophins is with the to of the receptor in Our of the of bestrophin from suggested by Bakall et al. B. Marknell T. S. Koisti M.J. Sandgren O. Li W. Bergen A.A. Andreasson S. T. K. Wadelius C. Genet. 1999; PubMed Scopus Google Scholar) and et al. (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) suggested transmembrane domains that to transmembrane domains and in The protein is proposed to have and et al. K. A. 2000; 15: PubMed Scopus Google Scholar) proposed transmembrane domains that to transmembrane domains and a domain that we is in the transmembrane domains and that the is and the is The by Bakall B. Marknell T. S. Koisti M.J. Sandgren O. Li W. Bergen A.A. Andreasson S. T. K. Wadelius C. Genet. 1999; PubMed Scopus Google Scholar), et al. (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), and et al. K. A. 2000; 15: PubMed Scopus Google Scholar) differ from in that their the RFP domain transmembrane in a However, the high of sequence in the RFP and to the M2 domain of LGACs that this is involved in the The of the RFP sequence is by the that mutations of the RFP sequence in non-functional channels. R. W. W. and H. C. the sequence of LGACs, the RFP sequence is at the end of a transmembrane by several In to the feature of bestrophins that to that they might be Ca2+-activated Cl- channels was the conserved domain the transmembrane Although this domain is to Ca2+ suggested to that it might be involved in Ca2+ it is very to and because of their high We were to for that were for the for and or is expressed. The that expressed were and The which is for that protein was expressed in and were in and and have high levels of both and whereas and have In in the of is high, but the of protein is This be the in was human bestrophin-1 is expressed in might that is playing the role in Xenopus that bestrophin-1 in Bestrophin vitelliform macular dystrophy is an early onset form of macular degeneration that has been to mutations in the human that for bestrophin-1 K. A. 2000; 15: PubMed Scopus Google Scholar). have been to be to mutations to to in the and transmembrane the the transmembrane and in the mutations have been in the of the protein or in the RFP mutations in the RFP domain produce a more that has not been of the that Best disease are conserved in the Xenopus bestrophins. Best disease is an dominant we the effect of two Best disease mutations on residues in the W93C and G299E mutations produce non-functional channels and the currents by wild type do these results that bestrophins are channels, they that the channels form and that the channels have a dominant negative effect on channel of bestrophin channels produce macular degeneration remains to be The that human bestrophin-1 is in retinal pigment has to the that bestrophin be the molecular of the Cl- channel in retinal pigment Although we have to a bestrophin in Xenopus that is more to human bestrophin-1 or we have not Our and results that in Xenopus play the role as human The high of expression in that this bestrophin be a of Ca2+-activated Cl- channels in or retinal In both and have Ca2+-activated Cl- currents C.R. D. J. Physiol. PubMed Scopus Google Scholar, T. P.R. J. 1994; PubMed Scopus Google Scholar, O. A. H. J. 1998; PubMed Google Scholar, S. B. J. Gen. Physiol. PubMed Scopus Google Scholar). currents have properties that are similar to the bestrophin currents we have has been suggested that the Ca2+-activated Cl- current in a role in the of the and in the effect of cell S. B. J. Gen. Physiol. PubMed Scopus Google Scholar). In Ca2+ activated Cl- currents play a role in and the light