PURIFICATION OF THE CIC-0 CHLORIDE CHANNEL FROM TORPEDO-CALIFORNICA ELECTROPLAX IDENTIFICATION OF A PHOSPHORYLATION SITE FOR CAMP-DEPENDENTPROTEIN-KINASE

The voltage-gated chloride channel (CIC-O) from the electric organ of Torpedo californica was purified by immunoaffinity chromatography, A p olyclonal antibody was shown to specifically recognize the CIC-O chann el (M(r) 85 000 - 90 000) in a Western blot of total membrane proteins . As monitored by immunoprecipitation, the formation of antibody-antig en complexes in solution strongly depends on the detergent composition . The highest yield of precipitated CIC-O was obtained from an incubat ion mixture containing both an anionic detergent, cholate or lauryl su lfate, and the zwitterionic detergent CHAPS, In contrast, immuno-preci pitation of CIC-O was largely reduced when cholate was exchanged for t he nonionic detergent Triton X-100, suggesting that the efficient form ation of immune-complexes is favored by negatively charged detergent, In initial immunopurification experiments, in addition to CIC-O a majo r contaminating polypeptide of M(r) similar to 115 000 was copurified, which represents the SITS-binding protein [Jentsch et al. (1989) Bioc hem. J. 261, 155]. Purification of CIC-O could be increased from about 35% up to 60% homogeneity when immunoaffinity chromatography was perf ormed in the presence of N-acetylglucosamine, Therefore the highly gly cosylated SITS-binding protein most likely interacts with the CIC-O pr otein via its carbohydrate parts, The purified CIC-O 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-O. Proteolytic digestion with en doproteinase GluC and HPLC-separation revealed two major phosphopeptid es, which could be identified by amino acid sequence analysis as diffe rent size fragments of the same consensus phosphorylation site, Compar ison of the peptide sequences with the deduced protein sequence of CIC -O [Jentsch et al, (1990) Nature 348, 510; O'Neill et al. (1991) Bioch em, Biophys. Acta 1129, 131] indicates serine 600 as the phosphorylate d residue. Therefore, our results provide strong evidence that CIC-O i s phosphorylated at this single site by PKA in vitro.