Covalent attachment of charybdotoxin to the beta-subunit of the high conductance Ca(2+)-activated K+ channel. Identification of the site of incorporation and implications for channel topology

• Published in: The Journal of biological chemistry Vol. 269; no. 37; pp. 23336 - 23341
• Main Authors: Knaus, H G, Eberhart, A, Kaczorowski, G J, Garcia, M L
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 16.09.1994
• Subjects: Amino Acid Sequence; Animals; Antibodies; Autoradiography; Calcium - metabolism; Cattle; Charybdotoxin; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Glycoproteins - metabolism; Lysine - metabolism; Molecular Sequence Data; Potassium Channels - chemistry; Potassium Channels - immunology; Potassium Channels - metabolism; Precipitin Tests; Scorpion Venoms - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)31658-7

Purified high conductance Ca(2+)-activated K+ (maxi-K) channels from bovine tracheal smooth muscle have been covalently labeled employing monoiodotyrosine charybdotoxin ([125I]ChTX) and different bifunctional cross-linking reagents. [125I]ChTX was specifically incorporated into the beta-subunit, which was thereafter isolated by size exclusion high performance liquid chromatography. Proteolytic fragments of the [125I]ChTX-labeled beta-subunit were generated by digestion with various endoproteinases. Glu-C or Asp-N cleavage yielded a glycosylated [125I]ChTX-labeled fragment of 13-14 kDa. A site-directed antiserum raised against residues 62-75 of the cloned beta-subunit of the maxi-K channel specifically recognizes the beta-subunit in immunostaining experiments and was capable of immunoprecipitating these ChTX-labeled peptides. Lys-C cleavage resulted in two fragments of 16 and 28 kDa, respectively, which were both precipitated by anti-beta (62-75). However, only the 28-kDa fragment was recognized by anti-beta(118-132) and shown to carry double the amount of N-linked carbohydrates. Taken together, these data restrict the site of covalent incorporation of ChTX into the beta-subunit exclusively at Lys69, confirm the predicted topology of this subunit, and indicate that both canonical N-linked glycosylation sites are occupied with complex carbohydrates of 5-6 kDa each. We propose that an extracellularly located portion of the beta-subunit is located within 7.7 A of the ChTX receptor site and could even participate in the formation of this receptor by close apposition of its extracellular domain with structural elements provided by the alpha-subunit.