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.
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.
Knaus
H G
HG
Institute for Biochemical Pharmacology, Innsbruck, Austria.
Eberhart
A
A
Kaczorowski
G J
GJ
Garcia
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D018999:Charybdotoxin; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D012604:Scorpion Venoms / Q000378:metabolism*
D000906:Antibodies; D003432:Cross-Linking Reagents; D006023:Glycoproteins; D015221:Potassium Channels; D012604:Scorpion Venoms; D018999:Charybdotoxin; D008239:Lysine; D002118:Calcium