Primary sequence and immunological characterization of beta -subunit of high conductance Ca super(2+)-activated K super(+) channel from smooth muscle

The charybdotoxin receptor, purified from bovine tracheal smooth muscle, consists of two subunits ( alpha and beta ) and, when reconstituted into planar lipid bilayers, forms functional high conductance Ca super(2+)-activated K super(+) channels. Amino acid sequence, obtained from proteolytic fragments of the beta -subunit, was used to design oligonucleotide probes with which cDNAs encoding this protein were isolated. The cDNAs encode a protein of 191 amino acids that contains two hydrophobic (putative transmembrane) domains and bears little sequence homology to subunits of other known ion channels. Site-directed antisera, raised against putative extracellular epitopes of this protein, specifically immunoprecipitated super(125)I-labeled Bolton-Hunter beta -subunit as well as [ super(125)I]charybdotoxin-cross-linked beta -subunit. Under nondenaturing conditions, however, these anti- beta sera immunoprecipitated a complex consisting of both the alpha - and beta -subunits. The data demonstrate that, in vivo, the high conductance Ca super(2+)-activated K super(+) channel exists as a multimer containing both alpha - and beta -subunits, and this cDNA represents the first beta -subunit of a potassium channel cloned to date. Furthermore, we demonstrate that the cloned protein is the subunit to which charybdotoxin is specifically and covalently incorporated when cross-linked to the channel.