Purification of the CIC-0 Chloride Channel fromTorpedo califomicaElectroplax Identification of a Phosphorylation Site for cAMP-Dependent Protein Kinase

Abstract

The voltage-gated chloride channel (CIC-0) from the electric organ of Torpedo californica was purified by immunoaffinity chromatography. A polyclonal antibody was shown to specifically recognize the CIC-0 channel (M(r) 85,000-90,000) in a Western blot of total membrane proteins. As monitored by immunoprecipitation, the formation of antibody-antigen complexes in solution strongly depends on the detergent composition. The highest yield of precipitated CIC-0 was obtained from an incubation mixture containing both an anionic detergent, cholate or lauryl sulfate, and the zwitterionic detergent CHAPS. In contrast, immuno-precipitation of CIC-0 was largely reduced when cholate was exchanged for the nonionic detergent Triton x-100, suggesting that the efficient formation of immuno-complexes is favored by negatively charged detergent. In initial immunopurification experiments, in addition to CIC-0 a major contaminating polypeptide of M(r) approximately 115,000 was copurified, which represents the SITS-binding protein [Jentsch et al. (1989) Biochem. J. 261, 155]. Purification of CIC-0 could be increased from about 35% up to 60% homogeneity when immunoaffinity chromatography was performed in the presence of N-acetylglucosamine. Therefore the highly glycosylated SITS-binding protein most likely interacts with the CIC-0 protein via its carbohydrate parts. The purified CIC-0 channel was found to be phosphorylated by PKA in vitro to a level of 0.35-0.4 mol of phosphate incorporated per mol of CIC-0. Proteolytic digestion with endoproteinase GluC and HPLC-separation revealed two major phosphopeptides, which could be identified by amino acid sequence analysis as different size fragments of the same consensus phosphorylation site. Comparison of the peptide sequences with the deduced protein sequence of CIC-0 [Jentsch et al. (1990) Nature 348, 510; O'Neill et al. (1991) Biochem. Biophys. Acta 1129, 131] indicates serine 600 as the phosphorylated residue. Therefore, our results provide strong evidence that CIC-0 is phosphorylated at this single site by PKA in vitro.

Purification and reconstitution of an outwardly rectified Cl− channel from tracheal epithelia

“…Previously, using PTX-induced ADP-ribosylation, we demonstrated that a G i protein copurified with the ORCC and that the ORCC was regulated by a G i protein in planar lipid bilayers (23). We also demonstrated that addition of PTX induced a shift in the I-V curve of the ORCC toward linearity (23). Here we tested whether the partly and highly purified 115-kDa protein preparations contained a G i protein.…”

“…Immunopurified Cl Ϫ channel proteins were reconstituted into liposomes as previously described (23). Concentrated protein samples (100 µl) were mixed with a phospholipid mixture [50:30:20 (wt/wt/wt) phosphatidylethanolaminephosphatidylserine-phosphatidylcholine] in the presence of 400 mM KCl, 0.5 mM MgCl 2 , and 5 mM Tris • HCl, pH 7.4.…”

“…Very good solubilization was achieved using the zwitterionic detergent CHAPS. The same detergent was successfully used for the solubilization of the ClC-0 Cl Ϫ channel from Torpedo californica electroplax (23). Another advantage of using CHAPS over nonionic detergents is that CHAPS more efficiently overcomes protein-protein interaction.…”