Loss of Hyperpolarization-activated Cl− Current in Salivary Acinar Cells from Clcn2 Knockout Mice
2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês
10.1074/jbc.m202900200
ISSN1083-351X
AutoresKeith Nehrke, Jorge Arreola, Ha‐Van Nguyen, Jodi Pilato, Linda Richardson, Gbolahan W. Okunade, Raymond B. Baggs, Gary E. Shull, James E. Melvin,
Tópico(s)Ion Channels and Receptors
ResumoClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl− current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl− current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of theClcn2 gene was generated. The resulting homozygousClcn2 −/− mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal inClcn2 −/− mice following in vivostimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2 −/−mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl− channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation. ClC-2 is localized to the apical membranes of secretory epithelia where it has been hypothesized to play a role in fluid secretion. Although ClC-2 is clearly the inwardly rectifying anion channel in several tissues, the molecular identity of the hyperpolarization-activated Cl− current in other organs, including the salivary gland, is currently unknown. To determine the nature of the hyperpolarization-activated Cl− current and to examine the role of ClC-2 in salivary gland function, a mouse line containing a targeted disruption of theClcn2 gene was generated. The resulting homozygousClcn2 −/− mice lacked detectable hyperpolarization-activated chloride currents in parotid acinar cells and, as described previously, displayed postnatal degeneration of the retina and testis. The magnitude and biophysical characteristics of the volume- and calcium-activated chloride currents in these cells were unaffected by the absence of ClC-2. Although ClC-2 appears to contribute to fluid secretion in some cell types, both the initial and sustained salivary flow rates were normal inClcn2 −/− mice following in vivostimulation with pilocarpine, a cholinergic agonist. In addition, the electrolytes and protein contents of the mature secretions were normal. Because ClC-2 has been postulated to contribute to cell volume control, we also examined regulatory volume decrease following cell swelling. However, parotid acinar cells from Clcn2 −/−mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl− channel in salivary acinar cells but is not essential for maximum chloride flux during stimulated secretion of saliva or acinar cell volume regulation. untranslated repeat reverse transcription-PCR cystic fibrosis transmembrane conductance regulator bovine serum albumin Spinner-modified minimal essential medium, RVD, regulatory volume decrease N-methyl-d-glucamine tetraethylammonium Molecular and functional studies have lead to the proposal that the inwardly rectifying Cl− channel in most, if not all, mammalian cells is ClC-2. Indeed, a null mutation in theClcn2 gene resulted in the loss of hyperpolarization-activated anion currents in Leydig and Sertoli cells (1Bosl M.R. Stein V. Hubner C. Zdebik A.A. Jordt S.E. Mukhopadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (256) Google Scholar). Inwardly rectifying Cl− currents have qualitatively similar properties in numerous tissues, nevertheless, unique activation kinetics are often observed in different cell types and in heterologous ClC-2 expression systems. For example, under identical experimental conditions, the chloride current generated by recombinant rat ClC-2 in HEK293 cells activates with a faster time course than the current in rat salivary acinar cells (2Park K. Arreola J. Begenisich T. Melvin J.E. J Membr. Biol. 1998; 163: 87-95Crossref PubMed Scopus (46) Google Scholar). Moreover, cAMP is an important regulator of recombinant human ClC-2 channel activity (3Tewari K.P. Malinowska D.H. Sherry A.M. Cuppoletti J. Am. J. Physiol. 2000; 279: C40-C50Crossref PubMed Google Scholar) and of hyperpolarization-activated Cl− currents in both choroid plexus (4Kibble J.D. Trezise A.E. Brown P.D. J. Physiol. 1996; 496: 69-80Crossref PubMed Scopus (39) Google Scholar) and human T84 colon cells (5Fritsch J. Edelman A. J. Physiol. 1996; 490: 115-128Crossref PubMed Scopus (81) Google Scholar); in contrast, cAMP sensitivity is not seen in salivary acinar cells (6Park K. Begenisich T. Melvin J.E. J. Membr. Biol. 2001; 182: 31-37Crossref PubMed Scopus (16) Google Scholar). One interpretation of these contrary results is differential expression of a regulatory subunit that modulates channel kinetics. Alternatively, splice variants of ClC-2 may alter the activation properties of this channel (7Chu S. Zeitlin P.L. Nucleic Acids Res. 1997; 25: 4153-4159Crossref PubMed Scopus (26) Google Scholar, 8Cid L.P. Niemeyer M.I. Ramirez A. Sepulveda F.V. Am. J. Physiol. 2000; 279: C1198-C1210Crossref PubMed Google Scholar). However, analysis of the currents in choroid plexus epithelial cells from ClC-2 knockout animals failed to reveal a loss of the hyperpolarization-activated Cl− conductance (9Speake T. Kajita H. Smith C.P. Brown P.D. J. Physiol. 2002; 539: 385-390Crossref PubMed Scopus (20) Google Scholar). These later results demonstrate that another novel gene encodes the inwardly rectifying Cl− current present in choroid plexus cells and raises the possibility that the hyperpolarization-activated Cl− channel in salivary gland cells and other cell types is not ClC-2. The physiological importance of some epithelial chloride channels has been revealed by gene mutation-inducing diseases such as cystic fibrosis (10Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-S. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5811) Google Scholar), Bartter's syndrome (11Simon D.B. Bindra R.S. Mansfield T.A. Nelson-Williams C. Mendonca E. Stone R. Schurman S. Nayir A. Alpay H. Bakkaloglu A. Rodriguez-Soriano J. Morales J.M. Sanjad S.A. Taylor C.M. Pilz D. Brem A. Trachtman H. Griswold W. Richard G.A. John E. Lifton R.P. Nat. Genet. 1997; 17: 171-178Crossref PubMed Scopus (753) Google Scholar), and nephrogenic diabetes insipidus (12Matsumura Y. Uchida S. Kondo Y. Miyazaki H., Ko, S.B. Hayama A. Morimoto T. Liu W. Arisawa M. Sasaki S. Marumo F. Nat. Genet. 1999; 21: 95-98Crossref PubMed Scopus (222) Google Scholar). In mice lacking ClC-2, degeneration of the retina and testis occurs, indicating that this chloride channel is required for the survival of cells that depend on epithelia forming blood-organ barriers (1Bosl M.R. Stein V. Hubner C. Zdebik A.A. Jordt S.E. Mukhopadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (256) Google Scholar). It is unclear whether this barrier function is related to the regulation of ClC-2 activity by extracellular pH (13Arreola J. Begenisich T. Melvin J.E. J. Physiol. (Lond.). 2002; 541: 103-112Crossref Scopus (47) Google Scholar, 14Jordt S.E. Jentsch T.J. EMBO J. 1997; 16: 1582-1592Crossref PubMed Scopus (202) Google Scholar) or cell swelling (15Gründer S. Thiemann A. Pusch M. Jentsch T.J. Nature. 1992; 360: 759-762Crossref PubMed Scopus (357) Google Scholar). The apical location of the ClC-2 channel in rat small intestine, renal, and airway epithelia further suggests that ClC-2 plays a role in regulating fluid and electrolyte movement in these tissues (16Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M., Li, C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 17Murray C.B. Chu S. Zeitlin P.L. Am. J. Physiol. 1996; 271: L829-L837PubMed Google Scholar). Indeed, antisense ClC-2 cDNA reduced native chloride current in the human intestinal cell line Caco-2 and significantly reduced Cl−-dependent secretion (16Mohammad-Panah R. Gyomorey K. Rommens J. Choudhury M., Li, C. Wang Y. Bear C.E. J. Biol. Chem. 2001; 276: 8306-8313Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Genetic analysis has provided important and sometimes surprising insights into the function of several chloride channels. Nevertheless, a clear understanding of the physiological significance of ClC-2 and other chloride channels in most epithelia remains to be determined. Functional analysis is complicated in native epithelial cells, because multiple types of chloride channels are typically present. Salivary gland acinar cells are no exception, expressing at least five distinct chloride conductances (18Zeng W. Lee M.G. Muallem S. J. Biol. Chem. 1997; 272: 32956-32965Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Arreola J. Park K. Melvin J.E. Begenisich T. J. Physiol. 1996; 490: 351-362Crossref PubMed Scopus (66) Google Scholar). The first of these to be characterized (20Iwatsuki N. Maruyama Y. Matsumoto O. Nishiyama A. Jpn. J. Physiol. 1985; 35: 933-944Crossref PubMed Scopus (56) Google Scholar) is activated by an increase in intracellular free [Ca2+] i (21Arreola J. Melvin J.E. Begenisich T. J. Gen. Physiol. 1996; 108: 35-47Crossref PubMed Scopus (137) Google Scholar). It is likely that the Ca2+-dependent Cl− channel is targeted to the apical membrane in parotid acinar cells as has been shown in pancreatic acinar cells (22Park M.K. Lomax R.B. Tepikin A.V. Petersen O.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10948-10953Crossref PubMed Scopus (89) Google Scholar). Because salivation is Ca2+-dependent (23Melvin J.E. Koek L. Zhang G.H. Am. J. Physiol. 1991; 261: G1043-G1050PubMed Google Scholar, 24Martinez J.R. Petersen O.H. Experientia (Basel). 1972; 28: 167-168Crossref PubMed Scopus (14) Google Scholar, 25Douglas W.W. Poisner A.M. J. Physiol. 1963; 165: 528-541Crossref PubMed Scopus (109) Google Scholar), the Ca2+-gated Cl− channel has been predicted to be the primary Cl− channel activated during stimulated secretion. Additional Cl− channels found in salivary gland cells include those that are volume-sensitive (26Arreola J. Melvin J.E. Begenisich T. J. Physiol. 1995; 484: 677-687Crossref PubMed Scopus (72) Google Scholar), cAMP-dependent (18Zeng W. Lee M.G. Muallem S. J. Biol. Chem. 1997; 272: 32956-32965Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), hyperpolarization-activated (2Park K. Arreola J. Begenisich T. Melvin J.E. J Membr. Biol. 1998; 163: 87-95Crossref PubMed Scopus (46) Google Scholar, 19Arreola J. Park K. Melvin J.E. Begenisich T. J. Physiol. 1996; 490: 351-362Crossref PubMed Scopus (66) Google Scholar), and channels with properties like ClC-0 (18Zeng W. Lee M.G. Muallem S. J. Biol. Chem. 1997; 272: 32956-32965Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The complexity created by the expression of multiple chloride channels in acinar cells indicates that gene knockout model systems will likely be required to unequivocally assign function to an individual channel. Therefore, we disrupted the Clcn2 gene to: 1) elucidate the molecular nature of the inwardly rectifying Cl− current in salivary acinar cells; 2) determine the role of ClC-2 in saliva secretion; and 3) examine whether ClC-2 is critical for cell volume regulation. Patch-clamp analysis of chloride currents in salivary gland acinar cells demonstrated the loss of inwardly rectifying current inClcn2 −/− mice. In contrast, no changes were observed in the calcium- or volume-activated chloride conductances. Despite suggestions that ClC-2 may be involved in volume regulation, acinar cells from Clcn2 −/− mice recovered cell volume following swelling by hypotonic shock as well as those from wild-type littermates. Furthermore, we show that the flow-rate of saliva secreted during in vivo stimulation, as well as the protein and electrolyte concentrations of the saliva, were comparable in wild-type and Clcn2 −/− mice. These data unequivocally identity Clcn2 as the gene that encodes for the inwardly rectifying Cl− channel in salivary acinar cells and demonstrate that the Cl− currents required for stimulated secretion of saliva are mediated by other channels such as the Ca2+- and/or volume-activated Cl−channels. A clone isolated from a 129/SVJ mouse genomic lambda library was used to construct a targeting vector with a neomycin-resistance (neo) gene as a positive selection marker and thymidine kinase gene as a negative selection marker. A 3.07-kb high fidelity PCR product obtained from a sub-cloned SstI fragment from the Clcn2 gene was inserted 3′ of the neo cassette, and a 2.3-kbEcoRI-BamHI fragment was inserted 5′ of the neo cassette (see Fig. 1 A). The neo cassette was designed to replace 1 kb of promoter and ∼500 nucleotides of 5′-UTR1 as well as exon 1 and most of exon 2 of the Clcn2 gene. However, after blunt-end cloning the 5′-fragment, a clone containing the wrong orientation of the 5′-arm was mistakenly identified and subsequently linearized and electroporated into KG ES cells. Southern blotting ofEcoRI-digested genomic DNA from targeted ES cell clones using a 1.3-kb outside probe (3′ of the sequence used to create the targeting vector, as indicated in Fig. 1 A) led to the identification of a recombinant clonal isolate exhibiting the predicted 11- to 7-kb shift in size (Fig. 1 C). Further characterization of this clone indicated that homologous recombination had taken place between the 3′-arm of the targeting vector and the genomic DNA, as well as between an unidentified segment of the 5′-arm of the targeting vector and a region slightly downstream of the 3′-end of the genomic copy of the 5′-arm (possibly due to the presence of long stretches of repetitive sequence in this area). Although this clone did not delete the promoter of the Clcn2 gene, the replacement of most of exons 1 and 2 with PGKneo was expected to and did, in fact, result in the loss of both the ClC-2 transcript and protein in the final homozygous animal, as determined by RT-PCR and Western analysis (Fig. 1 D). The generation of the anti-ClC2 antibody has been described previously (27Gyomorey K. Yeger H. Ackerley C. Garami E. Bear C.E. Am. J. Physiol. 2000; 279: C1787-C1794Crossref PubMed Google Scholar) and is a kind gift of C. Bear (The Hospital for Sick Children, Toronto). Amino acids 16–35 of rat ClC-2 contain the target sequence to which the antibody was raised. The targeted ES cell clone was injected into C57BL/6 blastocytes to generate chimeras that were backcrossed against the C57BL/6 strain. Germline transmission was assessed by Southern blotting, and heterozygous offspring were crossed to create the F2 animals used in the present study. In all cases, littermates were paired for each set of experiments. The general phenotype of ourClcn2 −/− strain was essentially indistinguishable from that reported recently by the Jentsch laboratory (1Bosl M.R. Stein V. Hubner C. Zdebik A.A. Jordt S.E. Mukhopadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (256) Google Scholar). For histological examination, adultClcn2 +/+ and Clcn2 −/−age- and sex-matched littermates (7–8 weeks of age) were anesthetized with 300 mg of chloral hydrate/kg of body weight (intraperitoneally) and the tissues were fixed by perfusion with 10% neutral buffered formalin, sectioned at 5 μm, and stained with hematoxylin and eosin. Whole cell currents were recorded at room temperature from freshly isolated single parotid acinar cells using the patch clamp technique (28Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15069) Google Scholar). An Axopatch 200 B amplifier (Axon Instruments Corp., Foster City, CA) was used to voltage clamp and record the resulting chloride currents. Voltage clamp protocols to activate channels were generated by pClamp 8 software (Axon Instruments Corp.). Chloride currents were filtered at 1 kHz using a low pass Bessel filter and digitized at 2 kHz. A glass pipette had a 2- to 4-MΩ resistance when filled with the internal solutions. To record hyperpolarization-activated chloride currents, cells were dialyzed with an internal solution containing (millimolar): TEA-Cl 140, EGTA 20, HEPES 20, pH 7.3 with TEA-OH. Calcium-dependent chloride channel currents were recorded from cells dialyzed with an internal solution containing (millimolar): NMDG-glutamate 80, NMDG-EGTA 50, CaCl2 30, HEPES 20, pH 7.3 with NMDG. The free calcium concentration of this solution was estimated to be 250 nm(WinMax 2, Stanford CA). Cells were bathed in an external hypertonic solution containing (millimolar): TEA-Cl 140, CaCl2 0.5,d-mannitol 100, HEPES 20, pH 7.3 with TEA-OH; volume-sensitive currents were activated by diluting this solution 20% with water and using the same internal solution as described above for recording hyperpolarization-activated chloride currents. Square pulses of 5 or 3 s were delivered every 7 s from a holding potential of 0 (hyperpolarization-activated and volume-sensitive currents) or −50 mV (calcium-dependent currents). Membrane potential was changed between −120 to +120 mV in 20-mV steps, and the resulting currents were recorded after 10 (hyperpolarization-activated) and 5 (volume-sensitive and calcium-dependent currents) min of dialysis. Junction potentials (4.5 mV) and leak currents were not corrected. Current-voltage relationships were constructed by plotting the absolute magnitude of the currents at the end of the pulse against the membrane potential. Parotid acinar cell clumps from adult (8–10 weeks old) Clcn2 +/+ andClcn2 −/− littermates were prepared by collagenase digestion as previously described (29Evans R.L. Bell S.M. Schultheis P.J. Shull G.E. Melvin J.E. J. Biol. Chem. 1999; 274: 29025-29030Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Briefly, mice were killed by exsanguination following exposure to CO2 gas. The parotid glands were quickly removed, trimmed of connective tissues, and finely minced in 7.5 ml of collagenase digestion medium (Eagle's minimal essential medium, Biofluids, Inc., Rockville, MD) containing 0.04 mg/ml collagenase P and 1% BSA. The minced glands were incubated at 37 °C in a shaker with continuous agitation (100 cycles/min) and under gas (95%O2 + 5%CO2). After the first 20-min interval the minced glands were dispersed by gentle pipetting (10 times) and centrifuged (210 × g for 15 s). The supernatant was discarded, and the pellet was resuspended in 7.5 ml of collagenase digestion medium for an additional 40 min with pipetting at 20-min intervals. The cells were then rinsed and harvested by centrifugation. Single cell preparations for electrophysiology utilized an initial 10-min digestion of the minced parotid tissue in 12.5 ml of trypsin digestion media (minimal essential medium, Spinner modification (SMEM), Biofluids, Inc.) containing 0.01% trypsin, 0.5 mm EDTA, and 1% BSA under 95%O2 + 5%CO2 gassing and while shaking (60 cycles/min). The cells were pelleted at 210 ×g for 15 s then washed with 10 ml of trypsin inhibitor solution (SMEM containing 0.2% trypsin inhibitor and 1% BSA). The cells were spun again and incubated in collagenase digestion solution as described above. Single cells were rinsed with BSA-free basal medium Eagle, selected by filtration through 53-μm nylon mesh, and attached to circular 5-mm polylysine-coated glass coverslides in a 37 °C incubator containing 95%O2 + 5%CO2. Cell volume was estimated using a Nikon Diaphot 200 microscope interfaced with an Axon Imaging Workbench System (Novato, CA). The dispersed acinar cells were loaded with the fluoroprobe calcein by incubation for 15 min at room temperature with 100% O2 in 2 μm calcein-AM (Molecular Probes, Eugene, OR). Dye-loaded cells were exposed to 490-nm light, and emitted fluorescence was measured at 530 nm. Changes in cell volume were monitored by measuring the fluorescence intensity of calcein within a delimited intracellular volume. Cell volume was expressed in arbitrary units as 1/normalized calcein fluorescence. Calcein-loaded acinar cell clumps were equilibrated in an isotonic physiological solution containing (in millimolar): 135 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.33 NaH2PO4, 20 Hepes, 10 glucose, 0.8 MgSO4, and 1.2 CaCl2, pH 7.4. Hypotonic challenge was induced by switching the perfusate to the above solution after diluting by 30% with water. Cell volume change was measured as described above. Regulatory volume decrease (RVD) was followed over the course of ∼300 s while the cells remained in the hypotonic solution, and the rate of volume recovery was calculated by determining the slope of the best-fit line following the switch to hypotonic media and maximum cell swelling. Some experiments used clotrimazole (Sigma Chemical Co.) at a final concentration of 1 μm present in all of the solutions, and others used zinc at a final concentration of 50 μm. Adult littermates Clcn2 +/+ andClcn2 −/− (7–8 weeks of age) were anesthetized with 300 mg of chloral hydrate/kg of body weight (intraperitoneally) and then stimulated with 10 mg of pilocarpine-HCl/kg of body weight (intraperitoneally). Whole saliva, primarily representing a combination of parotid and submandibular secretions, with a very minor component from sublingual and minor salivary, nasal, and tracheal glands, was collected from the lower cheek pouch by a suction device at intervals of 5, 10, and 15 min. The protein concentration of saliva was determined using the Bradford method. Total sodium and potassium contents in saliva samples were determined by atomic absorption using a PerkinElmer Life Sciences 3030 spectrophotometer. Saliva osmolality was measured using a Wescor 5500 vapor pressure osmometer, and chloride activity was determined using an Orion EA 940 expandable ion analyzer. A lambda genomic DNA library derived from 129/SVJ mice was screened using a probe specific for the Clcn2 gene. Genomic fragments from the resulting lambda clone were used to flank a positive (PGKneo) selection cassette in a vector designed to target the Clcn2 gene for disruption (Fig. 1 A). The construct was intended to replace a genomic segment that includes a portion of the Clcn2 promoter and 5′-UTR, as well as all of exon 1 and most of exon 2, with PGKneo. This strategy was expected to result in the inability to initiate transcription from the defunctClcn2 promoter in the transgenic strain, causing an absence of functional protein, thereby avoiding the possibility of a dominant negative effect caused by expression of a truncated protein. During the gene-targeting procedure, a construct with the upstream arm in the wrong orientation was mistakenly utilized. Electroporation of this construct into embryonic stem cells resulted in a single targeted cell line (out of ∼900 neomycin-resistant clones; Fig. 1 C) resulting from a hybrid homologous recombination/insertion event, as described below. After the mistake was recognized we learned that the promoter of another gene, encoding the RPB-17 protein, overlaps that ofClcn2 in rat (30Chu S. Blaisdell C.J. Liu M.Z. Zeitlin P.L. Am. J. Physiol. 1999; 276: L614-L624PubMed Google Scholar), as well as in mouse. 2K. Nehrke, unpublished observations. Because the correct construct would disrupt both genes, we proceeded to analyze the embryonic stem cell clone that we had identified. Analysis using both inside and outside probes as well as genomic PCR (data not shown) demonstrated that homologous recombination occurred between the 3′-arm of the targeting vector and the Clcn2gene. This was followed by a non-homologous insertion event in the upstream (backwardly oriented) arm (Fig. 1 B). The junction between the genomic and vector DNA was not mapped at the single nucleotide level due to a stretch of over 1500 nucleotides of up to 85% GC content, which precluded genomic PCR across that region. The final homozygous knockout strain was shown to lack ClC-2 protein by Western analysis (Fig. 1 D). In addition, RT-PCR with primers to the 5′- and 3′-ends of the transcript confirmed that ClC-2 message was not present in the knockout strain, whereas Northern analysis indicated that the RPB-17 mRNA, whose promoter overlaps that ofClcn2, but in the antisense orientation, was present at normal levels in the Clcn2 −/− animal (data not shown). The phenotype of the Clcn2 −/−strain generated in our laboratory is comparable to that reported recently (1Bosl M.R. Stein V. Hubner C. Zdebik A.A. Jordt S.E. Mukhopadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (256) Google Scholar). The mice appeared generally healthy and displayed normal behavior and body weight, but histological examination of semi-thin sections revealed abnormalities of the eye and testis. Unlike the wild-type eye (Fig. 2 A), the knockout exhibited post-natal degeneration of the retina, reflected by a gradual loss of photoreceptor cells and the outer nuclear layer, which was not present or remained as only a few cells adjacent to the inner nuclear layer (Fig. 2, B and C). Maturation of the testes was normal in wild-type mice (Fig.3 A) but was impaired in the knockout (Fig. 3 B). In Clcn2 −/−mice at the age of sexual maturity, the germ cell layers were missing, and no mature spermatozoa were present; in addition, there was hyperplasia of the Leydig cells and abnormal Sertoli cells were prominent and widespread (Fig. 3 B).Figure 3Histology of the testis. 5-μm sections of testis from a Clcn2 +/+ male (A) and Clcn2 −/− male (B) obtained 10 weeks after birth. The normal testicular architecture found in the +/+ mice is compromised in theClcn2 −/− animal, with missing germ cell layers (double-headed arrow in the +/+ control), abnormal Sertoli cells (small arrows) and a relative hyperplasia of the Leydig cells (large arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The homozygous knockout strain was used to assess the molecular nature of the inwardly rectifying Cl− current and to determine the role of ClC-2 in saliva gland function and fluid secretion from salivary acinar cells. The production of saliva is initiated by an increase in intracellular Ca2+ that opens Ca2+-dependent chloride channels on the apical membranes of the acinar cells. Anion fluxes in parotid acinar cells are mediated by at least five distinct chloride currents, namely, volume-sensitive, calcium-dependent, cAMP-activated, ClC-0-like, and hyperpolarization-activated channels (18Zeng W. Lee M.G. Muallem S. J. Biol. Chem. 1997; 272: 32956-32965Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Arreola J. Park K. Melvin J.E. Begenisich T. J. Physiol. 1996; 490: 351-362Crossref PubMed Scopus (66) Google Scholar). RT-PCR has demonstrated the presence of ClC-2 in parotid acini, and the characteristics of the hyperpolarization-activated chloride current in this cell type are quantitatively similar to that of the cloned ClC-2 channel (2Park K. Arreola J. Begenisich T. Melvin J.E. J Membr. Biol. 1998; 163: 87-95Crossref PubMed Scopus (46) Google Scholar). To unambiguously determine the molecular identity of the channel mediating this current, we performed patch clamp analysis on single parotid acinar cells isolated from wild-type andClcn2 −/− mice. The chloride currents recorded from wild-type parotid acinar cells using the whole cell configuration (Fig. 4, upper left trace) displayed inward rectification and time dependence, as has been observed previously (2Park K. Arreola J. Begenisich T. Melvin J.E. J Membr. Biol. 1998; 163: 87-95Crossref PubMed Scopus (46) Google Scholar, 19Arreola J. Park K. Melvin J.E. Begenisich T. J. Physiol. 1996; 490: 351-362Crossref PubMed Scopus (66) Google Scholar). To clearly monitor the hyperpolarization-activated chloride currents, it was necessary to eliminate the Ca2+-dependent and the volume-sensitive currents. This was accomplished using an internal pipette solution containing the calcium chelator EGTA and a hypertonic bath solution (see “Experimental Procedures”). Relative to acini from wild-type mice, currents for the acini ofClcn2 −/− mice decreased more than 10-fold in magnitude at the most negative potentials and exhibited no rectification (Fig. 4, upper right trace). The lower panels show the current-voltage (IV) relations of chloride currents for parotid acinar cells derived from multiple Clcn2 +/+ (left, n = 6) and Clcn2 −/− (right, n = 8) mice. A similar analysis of heterozygousClcn2 +/− mice revealed similar hyperpolarization-activated currents as present in wild-type acinar cells, suggesting that there is no dominant negative effect (data not shown). These results confirm that the ClC-2 channel is, in fact, responsible for the hyperpolarization-activated Cl−current in parotid acinar cells. Because the opening of the Ca2+-activated Cl−channel on
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