COVALENT ATTACHMENT OF CHARYBDOTOXIN TO THE BETA-SUBUNIT OF THE HIGH-CONDUCTANCE CA2-ACTIVATED K+ CHANNEL - IDENTIFICATION OF THE SITE OF INCORPORATION AND IMPLICATIONS FOR CHANNEL TOPOLOGY()

Purified high conductance Ca2+ activated K+ (maxi-K) channels hom bovi ne tracheal smooth muscle have been covalently labeled employing monoi odotyrosine charybdotoxin ([I-125]ChTX), and different bifunctional cr osslinking reagents. [I-125]ChTX was specifically incorpo rated into t he beta-subunit, which was thereafter isolated by size exclusion high performance liquid chromatography. Proteolytic fragments of the [I-125 ]ChTX-labeled beta-subunit were generated by digestion with various en doproteinases. Glu-C or Asp-N cleavage yielded a glycosylated [I-125]C hTX-labeled fragment of 13-14 kDa. A site-directed antiserum raised ag ainst 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. L ys-C cleavage resulted in two fragments of 16 and 28 kDa, respectively , which were both precipitated by anti-beta((62-75)). However, only th e 28-kDa fragment was recognized by anti-beta((118-132)) and shown to carry double the amount of N-linked carbohydrates. Taken together, the se data restrict the site of covalent incorporation of ChTX into the b eta-subunit exclusively at Lys(69), 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 prop ose that an extracellularly located portion of the beta-subunit is loc ated within 7.7 Angstrom of the ChTX receptor site and could even part icipate in the formation of this receptor by close apposition of its e xtracellular domain with structural elements provided by the alpha-sub unit.