Human ClC-3 Is Not the Swelling-activated Chloride Channel Involved in Cell Volume Regulation

Volume regulation is essential for normal cell function. A key component of the cells' response to volume changes is the activation of a channel, which elicits characteristic chloride currents (ICl, Swell). The molecular identity of this channel has been controversial. Most recently, ClC-3, a protein highly homologous to the ClC-4 and ClC-5 channel proteins, has been proposed as being responsible for ICl, Swell (1Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar). Subsequently, however, other reports have suggested that ClC-3 may generate chloride currents with characteristics clearly distinct from ICl, Swell. Significantly different tissue distributions for ClC-3 have also been reported, and it has been suggested that two isoforms of ClC-3 may be expressed with differing functions. In this study we generated a series of cell lines expressing variants of ClC-3 to rigorously address the question of whether or not ClC-3 is responsible for ICl, Swell. The data demonstrate that ClC-3 is not responsible for ICl, Swell and has no role in regulatory volume decrease, furthermore, ClC-3 is not activated by intracellular calcium and fails to elicit chloride currents under any conditions tested. Expression of ClC-3 was shown to be relatively tissue-specific, with high levels in the central nervous system and kidney, and in contrast to previous reports, is essentially absent from heart. This distribution is also inconsistent with the previous proposed role in cell volume regulation. Volume regulation is essential for normal cell function. A key component of the cells' response to volume changes is the activation of a channel, which elicits characteristic chloride currents (ICl, Swell). The molecular identity of this channel has been controversial. Most recently, ClC-3, a protein highly homologous to the ClC-4 and ClC-5 channel proteins, has been proposed as being responsible for ICl, Swell (1Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar). Subsequently, however, other reports have suggested that ClC-3 may generate chloride currents with characteristics clearly distinct from ICl, Swell. Significantly different tissue distributions for ClC-3 have also been reported, and it has been suggested that two isoforms of ClC-3 may be expressed with differing functions. In this study we generated a series of cell lines expressing variants of ClC-3 to rigorously address the question of whether or not ClC-3 is responsible for ICl, Swell. The data demonstrate that ClC-3 is not responsible for ICl, Swell and has no role in regulatory volume decrease, furthermore, ClC-3 is not activated by intracellular calcium and fails to elicit chloride currents under any conditions tested. Expression of ClC-3 was shown to be relatively tissue-specific, with high levels in the central nervous system and kidney, and in contrast to previous reports, is essentially absent from heart. This distribution is also inconsistent with the previous proposed role in cell volume regulation. regulatory volume decrease green fluorescence protein polymerase chain reaction enhanced green fluorescence protein polyacrylamide gel electrophoresis phosphate-buffered saline N-methyl-d-glucamine chloride The maintenance of a constant cell volume in the face of fluctuating intra- and extracellular osmolarity is essential for normal cell function. Following cell swelling upon exposure to hypotonic solution, animal cells restore their volume toward its original value by activation of channels and transporters in the plasma membrane: the loss of K+ and Cl− ions and organic osmolytes, followed by obligatory loss of water, leads to regulatory volume decrease (RVD)1 (2Lang F. Busch G.L. Ritter M. Volkl H. Waldegger S. Gulbins E. Haussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar). Although a cell swelling-activated chloride current (ICl, Swell) required for RVD has been carefully characterized in several cell types, the molecular identity of the channel has not yet been established (3Okada Y. Am. J. Physiol. 1997; 273: C755-C789Crossref PubMed Google Scholar, 4Strange K. Emma F. Jackson P.S. Am. J. Physiol. 1996; 270: C711-C730Crossref PubMed Google Scholar, 5Valverde M.A. Curr. Opin. Cell Biol. 1999; 11: 509-516Crossref PubMed Scopus (40) Google Scholar). This is primarily due to three experimental limitations. First, the current is ubiquitous, such that cell lines exhibiting little or no current necessary for expression cloning are not available. Second, no specific, high affinity blockers of the current are known. Third, the magnitude and the rate of activation of the currents are readily perturbed by both endogenous and exogenous factors making quantitative analysis difficult. The latter issue is illustrated by the fact that P-glycoprotein, proposed as a candidate for the cell swelling-activated chloride channel, was subsequently shown to be a regulator of endogenous channel activity (6Valverde M.A. Bond T.D. Hardy S.P. Taylor J.C. Higgins C.F. Altamirano J. Alvarez-Leefmans F.J. EMBO J. 1996; 15: 4460-4468Crossref PubMed Scopus (89) Google Scholar, 7Bond T.D. Valverde M.A. Higgins C.F. J. Physiol. (Lond.). 1998; 508: 333-340Crossref Scopus (40) Google Scholar), whereas another candidate, pICln, was also shown not to be the channel and its precise role is still uncertain (8Furst J. Bazzini C. Jakab M. Meyer G. Konig M. Gschwentner M. Ritter M. Schmarda A. Botta G. Benz R. Deetjen P. Paulmichl M. Pflugers Arch. 2000; 440: 100-115Crossref PubMed Google Scholar, 9Li C. Breton S. Morrison R. Cannon C.L. Emma F. Sanchez-Olea R. Bear C. Strange K. J. Gen. Physiol. 1998; 112: 727-736Crossref PubMed Scopus (40) Google Scholar). Another swelling-activated chloride channel expressed in many cell types, ClC-2, has also been suggested as contributing to RVD (10Xiong H. Li C. Garami E. Wang Y. Ramjeesingh M. Galley K. Bear C.E. J. Membr. Biol. 1999; 167: 215-221Crossref PubMed Scopus (55) Google Scholar). However ClC-2 generates a current with biophysical and pharmacological characteristics that differ significantly from ICl, Swell (11Grunder S. Thiemann A. Pusch M. Jentsch T.J. Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar, 12Jordt S.E. Jentsch T.J. EMBO J. 1997; 16: 1582-1592Crossref PubMed Scopus (206) Google Scholar, 13Bond T.D. Ambikapathy S. Mohammad S. Valverde M.A. J. Physiol. (Lond.). 1998; 511: 45-54Crossref Scopus (61) Google Scholar), and in a human intestinal epithelial cell line it has been shown that ClC-2 does not contribute to RVD (13Bond T.D. Ambikapathy S. Mohammad S. Valverde M.A. J. Physiol. (Lond.). 1998; 511: 45-54Crossref Scopus (61) Google Scholar). Recently, a series of studies has led to the suggestion that ClC-3 is the swelling-activated chloride channel responsible for ICl, Swell. The gene coding for ClC-3 was first cloned from rat by a homology-based cloning strategy; its predicted amino acid sequence is similar to other ClC channels (14Kawasaki M. Uchida S. Monkawa T. Miyawaki A. Mikoshiba K. Marumo F. Sasaki S. Neuron. 1994; 12: 597-604Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 15Kawasaki M. Suzuki M. Uchida S. Sasaki S. Marumo F. Neuron. 1995; 14: 1285-1291Abstract Full Text PDF PubMed Scopus (81) Google Scholar). The human cDNA was subsequently cloned and sequenced from fetal brain (16Borsani G. Rugarli E.I. Taglialatela M. Wong C. Ballabio A. Genomics. 1995; 27: 131-141Crossref PubMed Scopus (77) Google Scholar), and the guinea pig version was cloned and sequenced from cardiac myocytes (1Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar). Expression of gpClC-3 was reported to increase significantly ICl, Swell (1Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar, 17Duan D. Cowley S. Horowitz B. Hume J.R. J. Gen. Physiol. 1999; 113: 57-70Crossref PubMed Scopus (154) Google Scholar). This view was supported by antisense experiments where reduction of ClC-3 expression was reported to decrease ICl, Swell (18Wang L. Chen L. Jacob T.J. J. Physiol. (Lond.). 2000; 524: 63-75Crossref Scopus (113) Google Scholar). Subsequently, the role of ClC-3 in ICl, Swell has been challenged. Attempts to replicate experiments with gpClC-3 and hClC-3, either in Xenopus oocytes or in mammalian cell lines (HEK293 and NIH3T3), were unsuccessful (19Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In another study, expression of rClC-3 in CHO cells was reported to generate a Ca2+-sensitive chloride channel (15Kawasaki M. Suzuki M. Uchida S. Sasaki S. Marumo F. Neuron. 1995; 14: 1285-1291Abstract Full Text PDF PubMed Scopus (81) Google Scholar). Further complexity was introduced by the suggestion that short and long versions of ClC-3 could potentially be generated in vivo using two different translation initiation sites, identical except for an additional 58 amino acids at the N-terminal of the long version (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google Scholar). was reported that expression of the short and long versions in distinct currents that are and ICl, (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google Scholar), the of the currents is reported both as Cl− and Cl− X. K. Showalter L.A. Weinman S.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The tissue distribution of ClC-3 is also (14Kawasaki M. Uchida S. Monkawa T. Miyawaki A. Mikoshiba K. Marumo F. Sasaki S. Neuron. 1994; 12: 597-604Abstract Full Text PDF PubMed Scopus (218) Google reported coding for ClC-3 in brain and kidney, not in heart. In (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google reported high levels of expression of ClC-3 protein in the and C.F. D. Hume J.R. Horowitz B. Am. J. Physiol. 2000; PubMed Google high levels in heart. ClC-3 was reported in cells to be to the (18Wang L. Chen L. Jacob T.J. J. Physiol. (Lond.). 2000; 524: 63-75Crossref Scopus (113) Google Scholar), yet to the in (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google Scholar). the ClC-3 we generated cell lines expressing the long and short versions of with as in the long the short version of the swelling-activated currents significantly or cell volume regulation. ClC-3 generated no chloride currents in response to in The ClC-3 as the channel responsible for ICl, Swell and cell volume regulation. The original cDNA sequence coding for ClC-3 predicted a protein of amino acids short (14Kawasaki M. Uchida S. Monkawa T. Miyawaki A. Mikoshiba K. Marumo F. Sasaki S. Neuron. 1994; 12: 597-604Abstract Full Text PDF PubMed Scopus (218) Google Scholar). Subsequently, an was and a protein with an additional 58 amino acids at the was predicted long (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google Scholar). has been suggested that both versions may be expressed (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google Scholar). study the of hClC-3, we generated cell lines expressing both the short and long versions to at the has been reported that at the of ClC channels not K. J. T. J. Membr. Biol. 1998; PubMed Scopus Google Scholar). a for a cell line expressing with no was also cDNA were from (16Borsani G. Rugarli E.I. Taglialatela M. Wong C. Ballabio A. Genomics. 1995; 27: 131-141Crossref PubMed Scopus (77) Google Scholar). were from or generated from by and to generate a The at the was to a sequence and a of the The cDNA was as a the expression to generate the expression The cDNA was by generate the long the coding sequence was cloned from human by a reaction using followed by using for the was and at the The was as an which been by a of a of the short version The was generate the the system with an the with a was were generated by a cloning First, the was with an by using The from the was as a with and using two short to and the and The was a of T. and was the and gene as the generated cells J. R. J. Gen. PubMed Scopus Google cell expressing the hClC-3, and proteins, were generated by with generate a cell line expressing short a cells were with which and the at a of were for to and by cell The were and in fetal and for fluorescence and were for expression by A ClC-3, was by a to amino acids of the long version of amino acids of the short to with at a This was it is for ClC-3, and we that the does not with ClC-4 or ClC-5 in not from were using with the The were by and The were and using with a of The ClC-3, was from and was to of short The was a at with were with in phosphate-buffered saline with calcium and for at Cell were with with A for at The were with were by a a The was by with the line of an a The from in the cell was by using the line of a an or to were in of in for at The cells were with of in were by for at with of in and to in The reaction was by with of in and with The cells were by of of of protein was with of and at The was by and with of with of high in and with of The were by the with of for at and by Cell for were by cells with followed by and The was to the from were from from and in were at the were first with and in and in with and for using an were by the cell in a and with at for with was to a of and at for to of were with of and at for were by polyacrylamide at for in a The were to using a as by the with and for at the were at with and in the were three for with with the or as for at three for and the using the system currents were in cell of the as T.D. Valverde M.A. Higgins C.F. J. Physiol. (Lond.). 1998; 508: 333-340Crossref Scopus (40) Google Scholar). was the was an that a constant in the of the The extracellular to with The osmolarity was to with The extracellular hypotonic solution, to elicit swelling-activated the as the except that the osmolarity was with to with chloride were by with of the or and osmolarity was with to or for hypotonic or The intracellular was to with and osmolarity was to with are expressed as S.E. of were by was for or by the were and the for with in and osmolarity was to with with not cells were to a the of the were at were by a a and the was with a line of an The fluorescence was a the cells was and the fluorescence from a was the the fluorescence to hypotonic and by the of were F.J. Altamirano J. 1995; 27: Scopus (55) Google Scholar). The cells were for at with an and where was with and with a hypotonic solution, by reduction of The was and in volume and the of RVD was (6Valverde M.A. Bond T.D. Hardy S.P. Taylor J.C. Higgins C.F. Altamirano J. Alvarez-Leefmans F.J. EMBO J. 1996; 15: 4460-4468Crossref PubMed Scopus (89) Google Scholar, F.J. Altamirano J. 1995; 27: Scopus (55) Google Scholar). The fluorescence due to the in the cells expressing was in to the due to and was shown to be it could readily be for in the of the study of ClC-3 and its cell lines expressing short and long and short and long were generated in cells has been shown for other ClC channels that a does not K. J. T. J. Membr. Biol. 1998; PubMed Scopus Google Scholar). cell lines expressing is to to the plasma and to A. F. A. Jentsch T.J. S. A. 1998; PubMed Scopus Google Scholar, R. 1999; PubMed Scopus Google and short a were also with or ClC-3 and expression of the in cell that the with the at the the proteins, the cell lines were by of three the of a cell expressing short The in to the and the to the cell with The of the protein was to an in to the the and to a of intracellular the a of the protein was in a the of the the plasma A similar of was also for short the of and for of The cells shown were of the of the cells a and of several expressing protein not The data that a of the protein is to the plasma of the of the data not the that the protein is in the plasma demonstrate that the are in the plasma cells were with the Higgins C.F. EMBO J. 1999; PubMed Scopus Google Scholar). The were using and any in the protein were by and using In the of no were by the that the is not A of of the was to in with a plasma an intracellular to be the plasma the protein A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google was not that the does not the cell were with as a increase in of and was also were also with the expressing the ClC-3 protein not in the cell lines a of both the and is in the plasma established that the were in the plasma cell swelling-activated chloride currents were under the cell of the were at the of and by a from to in current in and exposure to a hypotonic at which the currents currents were in conditions except for cells expressing Following cell the a similar and and at in either the magnitude or rate of activation of ICl, Swell was the different currents were by of an of swelling-activated chloride channel M.A. Pflugers Arch. PubMed Scopus Google not chloride currents in ClC-3 and The chloride currents for the cell at are the of chloride current activation were from to exposure to to and are as a of the currents at The data were with a function. was the of activation of any of the cell lines The data shown are of two cell expressing short and for and the currents in and of exposure to are the the current for different the the in and in currents cell were except for ClC-5 expressing cells under conditions currents characteristics of ClC-5 currents also The of cells for cell line short long short short Although data with other ClC channels that a at the has no channel K. J. T. J. Membr. Biol. 1998; PubMed Scopus Google Scholar), we also that no in current magnitude or current characteristics were cells expressing ClC-3 and and studies (1Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google have suggested that the of ClC-3 of the current and changes its Expression of no in either the magnitude of swelling-activated chloride currents or its The of currents in cells expressing the protein not with the currents from cells expressing the for cell lines the Cl− In the rate of characteristics of swelling-activated currents in cells were by of any of the versions of tested. This is the fact that we that the were to the plasma and that similar expression of a generates characteristic data that ClC-3 as expressed has no channel the of ClC-3 in volume we RVD exposure to The of volume was by an from cells with the in of fluorescence are to changes in the of the which are changes in cell the of changes in cell volume (6Valverde M.A. Bond T.D. Hardy S.P. Taylor J.C. Higgins C.F. Altamirano J. Alvarez-Leefmans F.J. EMBO J. 1996; 15: 4460-4468Crossref PubMed Scopus (89) Google Scholar, F.J. Altamirano J. 1995; 27: Scopus (55) Google Scholar). a of both and the not The cells readily to an of and not of the an plasma not have an for the of it was not The cells that cell volume regulation no in RVD due to the expression of ClC-3 ClC-3 does not to be in cell volume regulation. is to ClC-3, and a current with ClC-5 expression has been in Xenopus oocytes and in cells (19Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). as a the in the cell line a similar of the protein was in the plasma in contrast to a current was in cells in conditions with an at Cl− This is similar to currents reported for and different in and from the endogenous swelling-activated currents in cells and in cells expressing of cells to a swelling-activated current in magnitude and from the endogenous currents of cells or cells and data demonstrate hClC-3, is under the conditions and elicits characteristic currents different from the cell swelling-activated currents in data a for the of currents expressing ClC-3 and also demonstrate that of a to ClC-5 has no current magnitude or The data the that ClC-3 is responsible for cell swelling-activated chloride and also that ClC-3 is a chloride channel at it is not under the conditions in this The other chloride channels that have been characterized in epithelial cells are activated by in intracellular cells expressing in response to of intracellular using the calcium This in both and cells currents with similar to epithelial chloride channels H. L. P. Nature. PubMed Scopus (154) Google Scholar, Am. J. Physiol. PubMed Google Scholar, S.A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). currents are in characteristics from the activated does not to a chloride volume decrease is a of cell (2Lang F. Busch G.L. Ritter M. Volkl H. Waldegger S. Gulbins E. Haussinger D. Physiol. Rev. 1998; 78: 247-306Crossref PubMed Scopus (1592) Google Scholar, Rev. 1995; PubMed Scopus Google Scholar). a ICl, Swell has been in cell K. Emma F. Jackson P.S. Am. J. Physiol. 1996; 270: C711-C730Crossref PubMed Google Scholar, 5Valverde M.A. Curr. Opin. Cell Biol. 1999; 11: 509-516Crossref PubMed Scopus (40) Google Scholar). any candidate protein for ICl, be to have a tissue expression of ClC-3 by in a of using an the of ClC-3 Although the the is not for ClC-3, of the two to protein that to is to that ClC-3 as in This may the predicted long and short both versions or may different high levels of ClC-3 were in and the central nervous for other and and and little or no was The from is in contrast to a previous C.F. D. Hume J.R. Horowitz B. Am. J. Physiol. 2000; PubMed Google this we also the with This a in cardiac tissue this was not by it a of of the the of ClC-3 in the In the distribution of ClC-3 is with a role of ClC-3 in and central nervous with a role in Cell volume regulation is an of and characteristic cell swelling-activated chloride currents an role in RVD in cell study, the molecular identity of the ICl, been Recently, ClC-3 has been proposed as a candidate for ICl, Swell with a role in cell volume regulation (1Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google Scholar, 17Duan D. Cowley S. Horowitz B. Hume J.R. J. Gen. Physiol. 1999; 113: 57-70Crossref PubMed Scopus (154) Google L. Chen L. Jacob T.J. J. Physiol. (Lond.). 2000; 524: 63-75Crossref Scopus (113) Google Scholar, C.F. D. Hume J.R. Horowitz B. Am. J. Physiol. 2000; PubMed Google Scholar). this has been (19Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google and by the of two a short and long version of ClC-3 (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google Scholar). In this study we have rigorously whether ClC-3 is responsible for ICl, Swell and that ClC-3 is responsible for ICl, a role in Cell lines were The of and extracellular were to demonstrate that a of ClC-3 was in the plasma the short long of ClC-3, at the of any ICl, Swell or cell volume regulation. of a ClC-3, that has been reported to both the and the of ICl, Swell (1Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (412) Google no the characteristics of the swelling-activated a cell lines generated in ClC-5 characteristic we generated ClC-3 and that the protein is expressed in a highly a with a role in RVD or in ICl, Swell. that the previous the role of ClC-3 is due to two experimental First, we have shown that the is not to ClC-3 and with other of similar molecular The of a ClC-3 has also to the tissue that ClC-3 is relatively in its being expressed primarily in and the Second, or mammalian cells swelling-activated chloride the magnitude of currents from cell to their magnitude is to such as and cell not a of cells is in a and and no of cells with high current is be in studies using we currents in cells expressing ClC-3 C.F. C. A. K. M. Valverde M.A. R. Scholar), it that this was a of experimental of the cells expression of ClC-3 ClC-3 is not responsible for ICl, Swell and has no role in is its Expression of the of is in or intracellular In contrast to a (18Wang L. Chen L. Jacob T.J. J. Physiol. (Lond.). 2000; 524: 63-75Crossref Scopus (113) Google Scholar), no ClC-3 was to the This previous was an of of A similar of expression was for a protein to be and in intracellular in the A. F. A. Jentsch T.J. S. A. 1998; PubMed Scopus Google Scholar, R. 1999; PubMed Scopus Google Scholar). This that ClC-3 may also have a role in intracellular expressed at the cell the long or short version of ClC-3 generated chloride currents ClC-5 as a and no current was in response to changes in intracellular calcium or cell A that short ClC-3 generates a current with a of Cl− X. K. Showalter L.A. Weinman S.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google is by a from the which that the short version generates currents with the (20Shimada K. Li X. Xu G. Nowak D.E. Showalter L.A. Weinman S.A. Am. J. Physiol. 2000; PubMed Google Scholar). no such currents generated by either the long or short the conditions or required to ClC-3 are are with ClC-3 being responsible for chloride currents at the plasma and are with an intracellular this was under Breiderhoff T. S. D. M. K. H. A. R. Jentsch T.J. Neuron. Full Text Full Text PDF PubMed Scopus Google that of the gene does not swelling-activated are to Jentsch for the ClC-5

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