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• W2036742255 abstract "Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b. Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b. Skeleton integrity requires that osteoclast-mediated bone resorption be intact and regulated (1Blair H.C. Zaidi M. Schlesinger P.H. Biochem. J. 2002; 364: 329-341Crossref PubMed Scopus (134) Google Scholar). Osteoclasts can remodel bone because these multinucleated cells secret sufficient acid into the resorption compartment to solubilize bone mineral (2Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (3004) Google Scholar, 3Schlesinger P. Mattsson J. Blair H. Miner. Electrolyte Metab. 1994; : 31-39PubMed Google Scholar), an alkaline salt that becomes increasingly soluble at pH values below 6 (4Cho G. Wu Y. Ackerman J.L. Science. 2003; : 1123-1127Crossref PubMed Scopus (265) Google Scholar, 5Neuman W.F. Neuman M.W. The Chemical Dynamics of Bone Mineral. The University of Chicago Press, Chicago, IL1958Google Scholar). The resorption compartment is delineated by a circumferential tightly adherent sealing zone (6Väänänen H.K. Zhao H. Mulari M. Halleen J.M. J. Cell Science. 2000; 113: 377-381Crossref PubMed Google Scholar). The osteoclast plasma membrane enclosed within the sealing zone differentiates into a highly specialized structure, the ruffled border. Across this membrane the cell actively transports HCl, which dissolves bone mineral and activates acid hydrolases required for bone resorption (7Baron R. Neff L. Louvard D. Courtnoy P. J.Cell Biol. 1985; : 2210-2222Crossref PubMed Scopus (592) Google Scholar, 8Silver I. Murrills R. Etherington D. Exp. Cell Res. 1988; 175: 266-276Crossref PubMed Scopus (725) Google Scholar). The transport of HCl occurs in two steps. An electrogenic ATP-dependent proton pump (9Blair H. Teitelbaum S. Ghiselli R. Gluck S. Science. 1989; 245: 855-857Crossref PubMed Scopus (727) Google Scholar, 10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 11Mattsson J.P. Schlesinger P.H. Keeling D.J. Teitelbaum S.L. Stone D.K. Xie X.-S. J. Biol. Chem. 1994; 269: 24979-24982Abstract Full Text PDF PubMed Google Scholar, 12Lee B.S. Holliday L.S. Ojikutu B. Krits I. Gluck S.L. Am. J. Physiol. 1996; : C382-C388Crossref PubMed Google Scholar), inserts H+ into the resorption compartment. Chloride ions follow passively through a parallel chloride conductance, short circuiting the electrogenic pump and allowing the massive HCl secretion necessary during bone resorption (10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar).We had previously identified a 62-kDa protein, p62, from avian osteoclast ruffled border that could be reconstituted to form a DNDS 2The abbreviations used are: DNDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonate; RT-PCR, reverse transcription-polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; GST, glutathione S-transferase; AP656, affinity-purified polyclonal antibody to CLIC-5b; E11, antibody to 31-kDa subunit of H+-ATPase.2The abbreviations used are: DNDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonate; RT-PCR, reverse transcription-polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; GST, glutathione S-transferase; AP656, affinity-purified polyclonal antibody to CLIC-5b; E11, antibody to 31-kDa subunit of H+-ATPase.-sensitive chloride channel (13Blair H. Schlesinger P. Biochem. Biophys. Res. Commun. 1990; 171: 920-925Crossref PubMed Scopus (37) Google Scholar). This protein is antigenically related to bovine CLIC-5b, a chloride channel of bovine kidney microsomal membranes (14Edwards J. Tulk B. Schlesinger P. J. Membr. Biol. 1998; 163: 119-127Crossref PubMed Scopus (54) Google Scholar). Expression of avian p62 in differentiating avian osteoclasts is coincident with the appearance of outwardly rectifying chloride conductance, valinomycin-independent acidification, and the ability of cells to resorb bone (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), all consistent with the hypothesis that p62 is the ruffled border chloride channel.Recently, a second protein, ClC-7, has been demonstrated to be important in bone resorption and proposed as being responsible for the ruffled border chloride conductance (16Kornak U. Kasper D. Bosl M. Kaiser E. Schweizer M. Schulz A. Friedrich W. Delling G. Jentsch T.J. Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar). ClC-7 is a member of the ClC family of proteins, several members of which are well known to function as plasma membrane chloride channels in a variety of cell types (17Jentsch T.J. Stein V. Weinreich F. Zdebik A.A. Physiol. Rev. 2002; 82: 503-568Crossref PubMed Scopus (1055) Google Scholar). The ClC channels are completely unrelated to the p64/CLIC family of proteins. Osteoclasts of animals in which ClC-7 was suppressed were abnormally elongated, had rudimentary ruffled borders and failed to resorb bone. Taken together, these data strongly support the identification of ClC-7 as a necessary component of the ruffled border acidification mechanism.Osteopetrosis results when osteoclasts are underperforming. Defects in osteoclast differentiation yield animals with dramatic dysfunction in the metabolism of bone including osteopetrosis (18Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Santos A. Van G. Itie A. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2834) Google Scholar, 19Yoshida H. Hayashi S. Kunisada T. Ogawa M. Nishikawa S. Okamura H. Sudo T. Shultz L.D. Nishikawa S. Nature. 1990; 345: 442-444Crossref PubMed Scopus (1506) Google Scholar). Many (∼50%) of the patients with malignant osteopetrosis have a defective proton pump (20Sobacchi C. Frattini A. Orchard P. Porras O. Tezcan I. Andolina M. Babul-Hirji R. Baric I. Canham N. Chitayat D. Dupuis-Girod S. Ellis I. Etzioni A. Fasth A. Fisher A. Gerritsen B. Gulino V. Horwitz E. Klamroth V. Lanino E. Mirolo M. Musio A. Matthijs G. Nonomaya S. Notarangelo L.D. Ochs H.D. Superti Furga A. Valiaho J. van Hove J.L.K. Vihinen M. Vujic D. Vezzoni P. Villa A. Hum. Mol. Genet. 2001; 10: 1767-1773Crossref PubMed Scopus (188) Google Scholar). A mild form of osteopetrosis has been associated with mutations in the chloride channel ClC-7 (16Kornak U. Kasper D. Bosl M. Kaiser E. Schweizer M. Schulz A. Friedrich W. Delling G. Jentsch T.J. Cell. 2001; 104: 205-215Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar). Studies on CD14 cells differentiated from patients with osteopetrosis indicated that biallelic mutations in ClC-7 produced acidic resorption compartments but were impaired in the removal of organic matrix (21Blair H.C. Borysenko C.W. Villa A. Schlesinger P.H. Kalla S.E. Yaroslavskiy B.B. Garcia-Palacios V. Oakley J.I. Orchard P.J. J. Bone Min. Res. 2004; 19: 1329-1338Crossref PubMed Scopus (31) Google Scholar). The ability of these cells to acidify the resorption compartment suggests that ClC-7 may play a role other than serving as the shunt chloride channel necessary for robust acidification.In addition to the proton pump and putative channel proteins, c-Src, the non-receptor-tyrosine kinase, is a key regulator of bone resorption (22Roodman D. Exp. Hematol. 1999; 27: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). c-Src is highly expressed in osteoclasts (23Horne W. Neff L. Chatterjee D. Lomri A. Levy J. Baron R. J. Cell Biol. 1992; 119: 1003-1013Crossref PubMed Scopus (208) Google Scholar) and concentrated at the ruffled border (24Tanaka S. Takahashi N. Udagawa N. Saski T. Fukui Y. Kurokawa T. Suda T. FEBS Lett. 1992; 313: 85-89Crossref PubMed Scopus (91) Google Scholar). c-Src negative transgenic mice develop osteopetrosis from decreased bone resorption (25Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1792) Google Scholar). Microscopically, osteoclasts from these mice were described to be elongated and have rudimentary or absent ruffled borders (26Boyce B. Yoneda T. Lowe C. Soriano P. Mundy G. J. Clin. Investig. 1992; 90: 1622-1627Crossref PubMed Scopus (516) Google Scholar). Therefore it appears that c-Src and its downstream signaling pathways support bone resorption by mature osteoclasts by allowing full development of the ruffled border (27Tanaka S. Amling M. Neff L. Peyman A. Uhlmann E. Levy J. Baron R. Nature. 1996; 383: 528-531Crossref PubMed Scopus (250) Google Scholar, 28Yoneda T. Lowe C. Lee C. Gutierrez G. Niewolna M. Williams P. Izbicka E. Uehara Y. Mundy G. J. Clin. Investig. 1993; 91: 2791-2795Crossref PubMed Scopus (97) Google Scholar, 29Sahni M. Zhou X. Bakiri L. Schlessinger J. Baron R. Levy J. J. Biol. Chem. 1996; 271: 3141-3147Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). A direct role for Src in support of osteoclast acid transport has not been determined, but it is clearly required to develop a functional ruffled border (2Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (3004) Google Scholar, 30Nakamura I. Rodan G.A. Duong L.T. J. Electron Microsc. 2003; 52: 527-533Crossref PubMed Scopus (25) Google Scholar).One mechanism by which c-Src could support the development of functional osteoclasts would be the targeting of proton pump, chloride channel, or both to the ruffled border (3Schlesinger P. Mattsson J. Blair H. Miner. Electrolyte Metab. 1994; : 31-39PubMed Google Scholar, 10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). p64, the bovine kidney homolog of osteoclast p62, has been shown to be regulated by the Src homolog, Fyn (31Edwards J.C. Kapadia S. J. Biol. Chem. 2000; 275: 31826-31832Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). When co-expressed with Fyn, p64 becomes tyrosine-phosphorylated and the tyrosine-phosphorylated p64 is a ligand for the SH2 domain of Fyn. Furthermore, co-expression of p64 with Fyn results in increased p64-associated chloride channel activity. These observations led us to speculate that, like p64, the osteoclast chloride channel might be a ligand for c-Src and that c-Src actions in osteoclasts could be mediated, at least in part, by that interaction.In this report, we separately disrupt expression of CLIC-5b and c-Src by avian osteoclasts differentiating in culture (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We present evidence that the avian ruffle border chloride channel, p62, is the avian form of CLIC-5b. We observe that suppression of CLIC-5b expression with antisense oligonucleotides reduces osteoclast bone resorption and decreases acidification in vesicles prepared from cultured osteoclasts. Based on the response to valinomycin, we conclude that the reduced acidification results from a decrease in chloride channel activity. These observations support our proposal that CLIC-5b is a ruffled border chloride channel and further confirm the importance of ruffled border chloride conductance in acidification and bone resorption. We investigate the binding of CLIC-5b to Src and demonstrate that the native protein is a ligand for both the SH2 and SH3 domains of Src. Finally, we investigate the effects of antisense suppression of c-Src on acid transport by isolated vesicles. Our data indicate that c-Src may be necessary for the functional expression of active chloride channel in proton pump containing membrane domains of differentiating osteoclasts.MATERIALS AND METHODSCulture of Osteoclasts and the Preparation of Membrane Vesicles—Osteoclasts and bone marrow cells were isolated from the long bones of calcium-deprived laying hens (Gallus domesticus), cultured and membranes prepared as described (10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 32Blair H. Kahn A. Crouch E. Jeffery J. Teitelbaum S. J. Cell Biol. 1986; 102: 1164-1172Crossref PubMed Scopus (218) Google Scholar). Membrane pellets were stored at –80 °C until analyzed.Assay of Vesicle Acidification—Acidification of membrane vesicles was as previously described (10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In brief, membrane vesicles formed in 140 mm KCl, 10 mm HEPES (pH 7.0) were diluted into the same buffer containing 3.3 μm acridine orange, 2.5 mm ATP, and other reagents as indicated. The reaction was started by adding 10 mm MgCl2 to the reaction mixture at 37 °C, and fluorescence was followed in an SPF500 spectrofluorometer (excitation 460 nm and emission 520 nm). At the end of each experiment 20 mm NH4Cl was added to determine the extent of intravesicular acidification. Protein concentration was determined using the BCA protein assay method (Pierce) and used to normalize results from different preparations. When studied in this manner the stimulated acidification of the reconstituted ruffled border membrane vesicles has an exponential time course. Therefore the quenching time course for each experiment was fitted to a single exponential using the Levenberg-Marquardt algorithm and Origin software. This analysis generated time constants for dequenching, t, and the size of the ATP-dependent exponential component of the total dequenching, F520time=0-F520time=∞. These values were employed to compare the effects of antisense suppression of CLIC-5b or Src on reconstituted vesicle acidification.Isolation of p64-related Sequences from Chicken—A commercially available chicken genomic library (Stratagene, La Jolla, CA) was screened at low stringency with a probe for the C-terminal portion of bovine p64, yielding a number of individual isolates. Restriction fragments hybridizing to the p64 probe were subcloned into plasmid vectors, random sequences generated and searched for homology with p64. One fragment corresponding to the C-terminal portion of p64 (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and a second corresponding to the middle of the p64 coding region were found. Oligonucleotides from those genomic sequences were used to generate cDNA from chicken osteoclast mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Northern blots of RNA from cultured differentiating osteoclasts was carried out as previously described (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), using the cloned PCR product as a probe. Several antisense oligonucleotides were selected from the sequence and assessed for ability to suppress expression of p62 in cultured cells. The most effective of these, 5′-CATCTGTCTTGACTTCTCC, was used in the following experiments. Phosphorothioate oligonucleotides containing this and matched complementary sense sequence was purchased from Midland Scientific (Midland, Texas).Antisense Treatment of Osteoclasts—Marrow cells from calcium-deprived hens were isolated as described and cultured in the presence of devitalized bone particles (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 32Blair H. Kahn A. Crouch E. Jeffery J. Teitelbaum S. J. Cell Biol. 1986; 102: 1164-1172Crossref PubMed Scopus (218) Google Scholar). After 24 h in culture, cells were exposed to phosphorothioate oligonucleotide using streptolysin-O permeabilization (33Chellaiah M. Fitzgerald C. Alvarez U. Hruska K. J. Biol. Chem. 1998; 273: 11908-11916Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 34Barry E. Gesek F. Friedman P. BioTechniques. 1993; 15: 1016-1018PubMed Google Scholar). Cells were washed in permeabilization buffer at 37 °C (137 mm NaCl, 100 mm PIPES at pH 7.4, 5.6 mm glucose, 2.7 mm KCl, 2.7 mm EGTA, 0.1% bovine serum albumin) and then exposed to 50 μm oligonucleotide in the same buffer supplemented with 1 mm ATP, 5 mm dithiothreitol and 0.5 units/ml streptolysin-O at 37 °C for 15 min. The cells were returned to growth medium for 2 h, washed an additional time with growth medium and returned to culture. The cells were harvested for analysis from 20 to 100 h after exposure to oligonucleotide. Avian c-Src expression was suppressed using antisense oligonucleotides as previously described (33Chellaiah M. Fitzgerald C. Alvarez U. Hruska K. J. Biol. Chem. 1998; 273: 11908-11916Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar).Bone Pitting Analysis—Marrow cells were plated directly onto ivory disks and exposed to oligonucleotide after 24 h exactly as described above. Forty-eight hours later the disks were stained using a toluidine blue-based two-stage procedure yielding the number of attached osteoclasts as well as the number of pits (35Dempster D. Murrills R. Horbert W. Arnett T. Bone and Min. Res. 1987; 2: 443-448Crossref PubMed Scopus (70) Google Scholar, 36Arnett T. Dempster D. Endocrinology. 1987; 120: 602-608Crossref PubMed Scopus (168) Google Scholar).Immunohistochemistry—Marrow cells were cultured onto thin microscope cover slips and exposed to oligonucleotide as described above. At 72 h in culture, the cells were fixed in 4% paraformaldehyde/PBS for 10 min, rinsed with PBS and permeabilized with 0.1% Triton X-100/PBS for 1 min. The cells were incubated overnight at 4 °C in 1:1000 dilution of AP656 (rabbit polyclonal antibody raised against an 18-amino acid peptide from CLIC-5b (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar)), washed and incubated overnight in 1:1000 mouse E11 (mouse monoclonal antibody specific for proton ATPase (37Bastani B. Ross F. Kopito R. Gluck S. Calcified Tissue Internat. 1996; 58: 332-336PubMed Google Scholar)), washed and incubated overnight with species-specific fluorescent secondary antibodies. Cover slips were mounted in Prolong antifade medium. Samples were viewed on a Zeiss LSM510 confocal microscope and z-stacks of cells were taken at 0.3-micron steps. These stacks were used to construct three-dimensional images that were analyzed using NOEsys, Research Systems, Inc. and final figures produced with Photoshop.Western Blotting—For detection of CLIC-5b, membrane preparations were analyzed by SDS-PAGE and probed with the AP656 antibody raised against a 18-amino acid peptide from CLIC-5b (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). For detection of Src, whole cell lysates were separated by SDS-PAGE, blotted, and probed with antibody raised against an avian Src (Upstate Biotechnology, Lake Placid, NY) using Supersignal detection reagents (Pierce).Immobilized Src Homology Domain Binding Studies—Regions encoding chicken Src SH2 domain (amino acids 145–240) and SH3 domain (amino acids 86–140) were generated by RT-PCR from chicken osteoclast RNA using standard methods, and inserted into plasmid pGEX-KG. This resulted in plasmids encoding glutathione S-transferase (GST) fused to the N terminus of either Src SH2 or SH3 domains as reported previously for Fyn SH2/SH3 (38Richard S. Yu D. Blumer K.J. Hausladen D. Olszowy M.W. Connelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Crossref PubMed Google Scholar). Fusion proteins were purified from bacteria and 1 mg of each was immobilized on 1 ml of Affi-Gel-15 beads (Bio-Rad). Membrane fractions from mature osteoclasts were solubilized in 150 mm NaCl, 10 mm Tris, pH 8.0, 1.4% n-octyl glucoside and insoluble debris removed by centrifugation at 100,000 × g for 1 h. For phosphatase treatment, 5 units of alkaline phosphatase (Promega) were added per mg of solubilized protein and incubated at room temperature for 30 min. Solubilized protein, 350 μg, was incubated with 20 μl of 50% suspension of Affi-Gel-15 beads bearing either GST (control beads), GST-SH2 fusion protein, or GST-SH3 fusion protein for 1 h at 4 °C. The beads were washed four times with solubilization buffer, suspended in Laemmli loading buffer, separated by SDS-PAGE and probed for CLIC-5b with affinity-purified antibody 656 as described previously (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar).Immunoprecipitation and in Vitro Kinase Assay—1 ml of crude 656 antiserum or of preimmune serum was immobilized on 2 ml of resin using the carbolink system (Pierce). Nonspecific binding sites were blocked by incubation with 1 mg/ml bovine serum albumin in PBS for 1 h. Membrane fractions from freshly isolated mature osteoclasts were solubilized in 200 mm sucrose, 10% glycerol, 10 mm Tris, pH 8.0, 1 mm EDTA, 1.4% n-octyl glucoside, 0.1 mm phenylmethylsulfonyl fluoride, and 0.1 mm sodium orthovanadate. Insoluble debris was removed by centrifugation and protein concentration determined. Overnight 300 μg of solubilized protein were incubated with 20 μlof 50% suspension of immune or preimmune beads at 4 °C with gentle mixing. Beads were washed with three changes of solubilization buffer followed by three changes of 200 mm LiCl, 200 mm Tris (pH 8), and finally one change of 20 mm HEPES (pH 7.4), 5 mm MgCl2, and 0.1 mm Na3VO4. The washed beads were then suspended in 10 μl of the same buffer and 1 μl of [γ-32P]ATP (3000 Ci/mm, 20 mCi/ml) was added for a 20-min incubation at room temperature. Fifteen microliters of 2× Laemmli loading buffer was added, the samples separated by SDS-PAGE and 32P-labeled proteins detected by autoradiography. As a positive control, 1 μl of purified human Src (Upstate Biotechnology, Lake Placid, NY) was added to an identical reaction mixture without the antibody beads.RESULTSProton Pump and Chloride Permeability in HCl Transport by Osteoclast Membrane Vesicles—Proton transport into membrane vesicles prepared from osteoclasts is electrogenic and therefore a compensating short circuit current is required for significant acidification (10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 11Mattsson J.P. Schlesinger P.H. Keeling D.J. Teitelbaum S.L. Stone D.K. Xie X.-S. J. Biol. Chem. 1994; 269: 24979-24982Abstract Full Text PDF PubMed Google Scholar). In these vesicles, endogenous sodium and potassium permeabilities are absent but chloride in the extravesicular medium permits robust acidification (10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 13Blair H. Schlesinger P. Biochem. Biophys. Res. Commun. 1990; 171: 920-925Crossref PubMed Scopus (37) Google Scholar). The chloride requirement can be overcome by introducing potassium permeability with valinomycin, a potassium ionophore. In the presence of equal intra- and extravesicular potassium concentration, valinomycin will clamp vesicle membrane potential at zero, thus permitting acidification of the vesicle interior to proceed at the maximum rate supported by the proton pump. We have previously shown that an imbalance of chloride permeability and proton transport by osteoclast membrane vesicles can be revealed by the effect of valinomycin on acidification (15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). If the chloride permeability of these vesicles is adequate to prevent generation of a membrane potential by the proton pump, valinomycin will have no additional effect on acidification. Conversely, if chloride permeability is limiting, valinomycin will collapse the membrane potential resulting from pump activity and thus enhance acidification.To study membrane potential-limited acidification in osteoclast vesicles we apply valinomycin and determine whether the rate or extent of acidification is increased. The kinetics of vesicle acidification was determined using an acridine orange technique as previously described (10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 11Mattsson J.P. Schlesinger P.H. Keeling D.J. Teitelbaum S.L. Stone D.K. Xie X.-S. J. Biol. Chem. 1994; 269: 24979-24982Abstract Full Text PDF PubMed Google Scholar). In this assay, acidification of intravesicular space leads to accumulation of acridine orange inside the vesicles and quenching of fluorescence (39Clerc S. Barenholz Y. Anal. Biochem. 1998; 259: 104-111Crossref PubMed Scopus (96) Google Scholar). Under these conditions acidification is an exponential approach to the steady state resulting from a balance of v-H+-ATPase activity and vesicle proton leak (40Grabe M. Oster G. J. Gen. Physiol. 2001; 117: 329-343Crossref PubMed Scopus (238) Google Scholar). In the presence of extravesicular chloride, vesicles acidify rapidly, as detected by quenching of acridine orange fluorescence, Fig. 1. Addition of valinomycin in the presence of chloride had minimal effect, indicating that these vesicles have sufficient shunt permeability to allow maximal acidification by the amount of proton pump present (10Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 15Schlesinger P. Blair H. Teitelbaum S. Edwards J. J. Biol. Chem. 1997; 272: 18636-18643Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Substitution for chloride with the impermeant anion, sulfate, or addition of the anion channel inhibitor, DNDS, reduced acidification. In either case electrogenic H+ transport by the pump generates a membrane potential, which increases prot" @default.
• W2036742255 created "2016-06-24" @default.
• W2036742255 creator A5003330445 @default.
• W2036742255 creator A5046706506 @default.
• W2036742255 creator A5056034482 @default.
• W2036742255 creator A5065471655 @default.
• W2036742255 date "2006-09-01" @default.
• W2036742255 modified "2023-10-15" @default.
• W2036742255 title "c-Src Control of Chloride Channel Support for Osteoclast HCl Transport and Bone Resorption" @default.
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• W2036742255 doi "https://doi.org/10.1074/jbc.m605865200" @default.
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