Regulation of Human CLC-3 Channels by Multifunctional Ca2+/Calmodulin-dependent Protein Kinase
Abstract
The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl−channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl− conductance (ICl,CaMKII), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of ICl,CaMKII. Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I− > Cl− to Cl− > I−. These results indicate that hCLC-3 encodes a Cl− channel that is regulated by CaMKII-dependent phosphorylation.AF172729 The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl−channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl− conductance (ICl,CaMKII), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of ICl,CaMKII. Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I− > Cl− to Cl− > I−. These results indicate that hCLC-3 encodes a Cl− channel that is regulated by CaMKII-dependent phosphorylation.AF172729 Ca2+/calmodulin-dependent protein kinase II reverse transcriptase-polymerase chain reaction polyacrylamide gel electrophoresis phosphate-buffered saline 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid 1,4-piperazinediethanesulfonic acid 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid cystic fibrosis Gating of the chloride channel, CLC-3, which is expressed in brain and chloride (Cl−) secretory epithelial tissues has remained controversial since its initial identification and characterization (1Kawasaki 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). The 760-amino acid protein encoded by theClc-3 gene was originally cloned from rat kidney and showed abundant expression in rat brain, most notably in the olfactory bulb, hippocampus, and cerebellum (1Kawasaki 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). When expressed in a stably transfected cell line, the rat kidney isoform of the channel showed basal activation, inhibition by phorbol esters, and Ca2+sensitivity (2Kawasaki M. Suzuki M. Uchida S. Sasaki S. Marumo F. Neuron. 1995; 14: 1285-1291Abstract Full Text PDF PubMed Scopus (81) Google Scholar). Duan and colleagues (3Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (413) Google Scholar, 4Duan D. Cowley S. Horowitz B. Hume J.R. J. Gen. Physiol. 1999; 113: 57-70Crossref PubMed Scopus (154) Google Scholar) characterized the functional expression of a cardiac clone of guinea pigclc-3, which when expressed in NIH 3T3 cells resulted in a large basally active Cl− conductance that was activated by an increase in cell volume, inhibited by phorbol esters and exhibited biophysical properties at the single channel level identical to the swelling activated current in native cardiac myocytes. Shimada and colleagues (5Shimada K. Li X. Xu G. Nowak D. Showalter L. Weinman S. Am. J. Physiol. Gastroint. Liver Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar) examined rat hepatocytes for Clc-3 expression and found that mRNA for two different isoforms was present; a short form corresponding to the guinea pig clone, and a long form containing a putative 58-amino acid addition at the N terminus of the protein. Both isoforms gave rise to current expression with identical selectivity when transiently expressed in CHO-K1 cells; however, they differed in the degree of outward rectification and voltage-dependent inactivation (5Shimada K. Li X. Xu G. Nowak D. Showalter L. Weinman S. Am. J. Physiol. Gastroint. Liver Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar). Adding a further dimension to the controversy, Friedrich and colleagues (6Friedrich T. Breiderhoff T. Jentsch T. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) report in a mutational analysis of Clc-4 and Clc-5 that they were unable to detect currents upon Clc-3 expression in Xenopus oocytes or in transfected HEK293 cells. Recent evidence from the studies of Stobrawa et al. (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) in transgenic mice with disrupted Clc-3 demonstrates that the chloride channel is broadly expressed and present in endosomal compartments and neuronal theClc-3 mice remained they remained for the most which Stobrawa and colleagues (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) demonstrated in the mice were the of the the of (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). The of both the and was to of in mice Stobrawa and colleagues (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) to that Clc-3 an anion which for the which the to membrane (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). The that Clc-3 is an chloride channel expressed in both endosomal that in with the plasma membrane at a level which is upon in the tissues in which is expression of Clc-3 at upon a with the plasma membrane and membrane been in cells and secretory regulate cellular volume, and The multifunctional Ca2+/calmodulin-dependent protein kinase II is a of is expressed in the brain, and is found in cell of by CaMKII has been shown in cells from the human colonic tumor cell line, Am. J. Physiol. PubMed Google Scholar, J.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Physiol. PubMed Scopus Google Scholar, D. L. A. Am. J. Physiol. PubMed Google H. L. Nature. PubMed Scopus (154) Google H. Neuron. Full Text PDF PubMed Scopus Google human F. J. Biol. 1994; PubMed Scopus Google epithelial cells T. Am. J. Physiol. PubMed Google and cystic epithelial cells K. J. 1995; PubMed Scopus Google Scholar). The of the channel or the CaMKII-dependent conductance has remained the current in the to the for expressed the expression of the CaMKII-activated Cl− conductance in native and the presence of CaMKII in the acid in the present was to the that hCLC-3 is a regulated by CaMKII. this cloned a full-length hCLC-3 gene from cells. gene was identical to the rat long form by Shimada and colleagues (5Shimada K. Li X. Xu G. Nowak D. Showalter L. Weinman S. Am. J. Physiol. Gastroint. Liver Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar). experiments demonstrated that at a of hCLC-3 is expressed at the of the stably transfected cells. showed that an increase in the of CLC-3 from a to was for current expression in the stably transfected cells demonstrated in membrane We the whole cell to the and selectivity of hCLC-3 stably expressed in a human kidney cell line HEK293 cells. show that the functional expression of the hCLC-3 Cl− conductance which is regulated by CaMKII with properties of endogenous in secretory A mutation in the putative pore a in anion selectivity from the I− > > Cl− to Cl− > > I− further that the transfected channel was for the current in the stably transfected cell hCLC-3 was cloned from a human colonic tumor cell line by were from the hCLC-3 gene cloned from a human by et al. G. M. C. A. 1995; PubMed Scopus Google Scholar). The was into vector and A of the et al. G. M. C. A. 1995; PubMed Scopus Google Scholar) with the clone which report two two acid and in the et al. a mutation at in the et G. M. C. A. 1995; PubMed Scopus Google Scholar) clone to in the In to the cloned the full-length hCLC-3 from The was with the hCLC-3 cloned from cells The full-length hCLC-3 was into the vector and transfected into tsA cells transfected cell were at and at The expression of hCLC-3 was by and the of the clone in this was by with The glycine at 280 was to a glutamic acid the with hCLC-3 were at The mutation was by hCLC-3 was with vector into tsA cells at a transfected cells were by expression of protein. cell were at were and by to were to hCLC-3 in The to an and to with an and glutamic acid in the a was to or The was for and for The of the was by whole cell from HEK293 cell expressing human rat and human of K. A. and C. cells were in in the presence of a inhibitor The membrane and were by of the whole cell at for at was with of and and to for the protein was with of for at protein was by and to were at with at a or at a and with to at a for was for the by Cells were in of and containing the of was with to with of with of A protein was to for in containing and of the to or with of was for the The membrane was with at a The tsA cells stably transfected with hCLC-3 were to in a The cells were with and into an in was to the cells at a of at for a The cells were and the was the of was and for the cells were with for and in with protein from the cell was with of or with of A and in of containing The protein was for The membrane was with at a and at cells were from rat and colonic was from membrane by and with by et al. C. J. 1994; PubMed Scopus Google Scholar). protein to or with of was for Cells were with Cells were with or for at by in for at and with for at Cells were with at a or protein at a for at with and with or for at Cells to the were The were a and cell patch clamp experiments were both and hCLC-3 transfected tsA cells. were to J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Physiol. PubMed Scopus Google Scholar). were a of from to in from a of were in and at The and was In of the experiments, was in the the in of in the The and rat brain CaMKII was in The CaMKII was previously J.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The kinase was introduced into the cell via the A of the inhibitor autocamtide-2-related inhibitory A. S. T. H. 1995; PubMed Scopus Google Scholar, A. K. S. T. H. PubMed Scopus Google Scholar) in was to with The of was to with cell were an patch clamp The a The the and for the in the by K. J. Physiol. 2000; PubMed Scopus Google Scholar). The of the was a The in the experiments was experiments were at expressed with the of experiments in The of the results was The full-length hCLC-3 was cloned from the human colonic tumor cell line The putative hCLC-3 protein is acid in with the long form Clc-3 cloned from rat hepatocytes (5Shimada K. Li X. Xu G. Nowak D. Showalter L. Weinman S. Am. J. Physiol. Gastroint. Liver Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar). The at the N terminus from guinea pig by Duan et al. (3Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (413) Google Scholar). The 760-amino acid with both guinea pig and rat short form of the putative hCLC-3 acid for of The of resulted in putative CaMKII in hCLC-3 at We two corresponding to the or of The of the was by whole cell from cell expressing and The corresponding for of the chloride channel was and The were to expression of hCLC-3 in stably transfected tsA cells. Both a protein from whole cell with a from to A single of was from the whole cell with which was with the for hCLC-3 of The was in the membrane from the that CLC-3 has been to in cells from the (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google experiments to hCLC-3 was expressed at the cell Cells stably transfected with hCLC-3 were with or and with A single protein was at and was in the that a of hCLC-3 expression is at the cell of endogenous hCLC-3 in the human colonic epithelial cell line, T84, and in the cell line was to by protein expression in cells was by with by with endogenous hCLC-3 in both the and cell a of protein in the and of rat and colonic were by protein was by and with from to was in the membrane The in for CLC-3 is with analysis of membrane from mice (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). CLC-3 a of an level of CLC-3 in was characterized by a of which of the of CLC-3 was in and the stably transfected tsA cells in the level of and membrane We that an increase in the expression of CLC-3 to Cells were and with to or to the in the a of from the to the plasma membrane in both cell a cells were the with a protein, protein demonstrates that remained in the and of cells were with an a protein, protein were with an a to and with for at cell patch clamp experiments were in to the functional expression of were in Cl− was the and the Cl− was current was a in clamp experiments were cells that been in for The CaMKII was introduced into the cells via the patch has been previously J.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Physiol. PubMed Scopus Google Scholar). currents the of the whole cell were in cells and in over the current in studies was for the initial in current the into the current was to a over in the presence of CaMKII from tsA cells stably transfected with hCLC-3 or vector is in A. was a increase in Cl− current over in of tsA cells transfected with the hCLC-3 gene CaMKII current density at was which is that in cells transfected with vector The of was with a in of The chloride channel inhibited and outward at and at > In to that the current was a of experiments were and in D. The of increase current density over that in experiments in which kinase was in the > When the autocamtide-2-related inhibitory a CaMKII specific inhibitor A. S. T. H. 1995; PubMed Scopus Google Scholar) was in the with the CaMKII, current was in cells the that CaMKII CLC-3 an of from experiments were in the presence of and the kinase in the The in experiments was different from that when were to with and In to hCLC-3 was expressed a functional channel and a which of an endogenous channel, introduced a mutation in the that encodes a glutamic acid at 280 for a glycine in a region which is conserved the ClC channel The anion of was for both and hCLC-3 transfected tsA cells in In experiments, of the Cl− was by Cells were to a to of the Cl− to the The for anion was The selectivity for from and the was I− > > Cl− for and Cl− > > I− for the In addition to the of the I− to Cl− the showed a in outward rectification in the from the cells of the anion of the CaMKII-dependent anion conductance in tsA cells stably transfected with hCLC-3 and in tsA cells transiently transfected with anion was in tsA cells expressing hCLC-3 and tsA cells transiently expressing the hCLC-3 of from currents in cells. of cells is in the was changed from a Cl− to containing of the is I− or were from the in a anion was in tsA cells expressing hCLC-3 and tsA cells transiently expressing the hCLC-3 of from currents in cells. of cells is in the was changed from a Cl− to containing of the is I− or were from the We membrane experiments to the increase in cell in the hCLC-3 stably transfected tsA cells. In experiments examined the of a membrane conductance increase in the presence of the from studies and in We a increase in in the presence of CaMKII over that in the of the kinase and in membrane were in both and were with membrane conductance These experiments to that a was for conductance in the tsA cells. These experiments the that to a conductance in the presence of the kinase in secretory cells expressing In to hCLC-3 is activated by in cell volume, Cl− current in the vector transfected to that of tsA cells. In the presence of the current was The Cl− in the was and the was that the current was The current density of both the basal and current from the cells was different from currents from the hCLC-3 stably transfected tsA cells in B. CaMKII introduced of two hCLC-3 into the hCLC-3 transfected tsA or cells to the in both CaMKII and Cl− current In the presence of from tsA cells that express hCLC-3 in current density with the presence of in the inhibited current by the kinase The anion current was inhibited by The results were from cells that express endogenous the inhibition of the CaMKII-dependent current by was via of the and the the in the two was In the presence of kinase by These that the inhibition is its with the We present in this which demonstrates that the expression of the Cl− channel hCLC-3 is in a and the plasma The of the protein from the to plasma membrane to in Cl− secretory cells in a in native Cl− secretory is to membrane CLC-3 current is upon a CaMKII-dependent is with in membrane and is with in cellular experiments in the stably transfected tsA cells showed a of CLC-3 expression by experiments and they the that expression via with the plasma membrane in secretory results with of Stobrawa et al. (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) which the expression of CLC-3 to endosomal compartments and neuronal The function of the protein of the Clc-3 gene cloned originally the and epithelial cell (1Kawasaki 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, M. Suzuki M. Uchida S. Sasaki S. Marumo F. Neuron. 1995; 14: 1285-1291Abstract Full Text PDF PubMed Scopus (81) Google Scholar) has been the of a of and colleagues (1Kawasaki 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) demonstrated current of the rat in Xenopus oocytes and found that the channel currents were inhibited by protein kinase C. The abundant expression of the in brain and its by protein kinase a for the conductance in the and of long of the gene in mammalian cell in a by et al. (2Kawasaki M. Suzuki M. Uchida S. Sasaki S. Marumo F. Neuron. 1995; 14: 1285-1291Abstract Full Text PDF PubMed Scopus (81) Google Scholar) single channel to show that an increase in channel In the guinea pig has been a Cl− channel (3Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (413) Google Scholar, 4Duan D. Cowley S. Horowitz B. Hume J.R. J. Gen. Physiol. 1999; 113: 57-70Crossref PubMed Scopus (154) Google Scholar). The Cl− conductance with expression is characterized by large basal and to properties which by the endogenous and rat Cl− conductance S. A. S. J. Gen. Physiol. PubMed Scopus Google Scholar). the human isoform of CLC-3 from the rat and guinea pig isoform by the addition of in the to that with a different functional Shimada and colleagues (5Shimada K. Li X. Xu G. Nowak D. Showalter L. Weinman S. Am. J. Physiol. Gastroint. Liver Physiol. 2000; 279: G268-G276Crossref PubMed Google Scholar) shown that is expressed two isoforms corresponding to the and corresponding to the hCLC-3 which report were to that both isoforms express functional Cl− in outward rectification and presence of inactivation at Recent studies from and colleagues (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) that CLC-3 is expressed in neuronal endosomal and compartments an anion for the studies the characterization of the CaMKII-activated anion conductance in shown that is Am. J. Physiol. PubMed Google Scholar, J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Physiol. PubMed Scopus Google Scholar, H. L. Nature. PubMed Scopus (154) Google Scholar, H. Neuron. 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Am. J. Physiol. PubMed Google Scholar, S. A. PubMed Scopus Google Scholar, Nature. 1997; 390: PubMed Scopus Google Scholar). that is by hCLC-3 and to of an channel endogenous to tsA a mutation in hCLC-3 was and transiently expressed in tsA cells. The anion for the was changed to Cl− > > the that the human isoform of CLC-3 for a CaMKII-activated mutational analysis of the region of in and colleagues Nature. 1997; 390: PubMed Scopus Google Scholar) to a putative pore region at the of a motif that is conserved the ClC channel family Nature. 1997; 390: PubMed Scopus Google Scholar). the mutation in channel to and the anion from Cl− > > I− to I− > Cl− > Nature. 1997; 390: PubMed Scopus Google Scholar). The mutation in the I− to Cl− in the that is to the however, a in the properties the of the two CLC-3 with and and form a of the gene studies of and show that they plasma membrane currents in that they characterized by outward Friedrich and colleagues (6Friedrich T. Breiderhoff T. Jentsch T. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) that they were unable to basally active CLC-3 currents in oocytes or transfected cells in to the currents by (1Kawasaki 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, M. Suzuki M. Uchida S. Sasaki S. Marumo F. Neuron. 1995; 14: 1285-1291Abstract Full Text PDF PubMed Scopus (81) Google Scholar) and Duan et al. (3Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (413) Google Scholar, 4Duan D. Cowley S. Horowitz B. Hume J.R. J. Gen. Physiol. 1999; 113: 57-70Crossref PubMed Scopus (154) Google Scholar). for the of currents in the Friedrich et al. (3Duan D. Winter C. Cowley S. Hume J.R. Horowitz B. Nature. 1997; 390: 417-421Crossref PubMed Scopus (413) Google Scholar, 4Duan D. Cowley S. Horowitz B. Hume J.R. J. Gen. Physiol. 1999; 113: 57-70Crossref PubMed Scopus (154) Google Scholar) studies that channel is We examined the of hCLC-3 by cell We in stably transfected tsA cells an to The current of the from vector and hCLC-3 transfected tsA cells were that of with an of In to further the hCLC-3 and examined the functional of specific to CLC-3, which were introduced via the into the stably transfected tsA cells cells expressing ICl,CaMKII. In both cell was abolished in the presence of the In both remained The that and by the protein. The of current inhibition by to the channel or protein of or a that is for results that the of hCLC-3 is by cell is the to is that CaMKII the channel or a is that CaMKII-dependent increase of the movement of from a to the plasma or the of containing hCLC-3 protein to the The was by experiments the of in the of ICl,CaMKII. CLC-3 has a in the an et al. (1Kawasaki 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) of Clc-3 expression in rat brain G. M. C. A. 1995; PubMed Scopus Google Scholar). The cellular and of CLC-3 in the is in the mice which showed a of the (7Stobrawa S. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A. Boesl M. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). In this shown that is expression of hCLC-3 in cells in function Cl− secretory epithelial cells and cells of the of hCLC-3 to of epithelial cells the and its expression in the that the channel an for in of an Cl− conductance in the epithelial secretory cells in has long been a in the in the The of which the of secretory in the CaMKII-dependent Cl− conductance upon identification of the channel at the the of CLC-3 in the plasma membrane and the of the channel to to in We J. R. for