PRIMARY SEQUENCE AND IMMUNOLOGICAL CHARACTERIZATION OF BETA-SUBUNIT OF HIGH-CONDUCTANCE CA2-ACTIVATED K+ CHANNEL FROM SMOOTH-MUSCLE()

The charybdotoxin receptor, purified from bovine tracheal smooth muscl e, consists of two subunits (alpha and beta) and, when reconstituted i nto planar lipid bilayers, forms functional high conductance Ca2+-acti vated K+ channels. Amino acid sequence, obtained from proteolytic frag ments of the beta-subunit, was used to design oligonucleotide probes w ith which cDNAs encoding this protein were isolated. The cDNAs encode a protein of 191 amino acids that contains two hydrophobic (putative t ransmembrane) domains and bears little sequence homology to subunits o f other known ion channels. Site-directed antisera, raised against put ative extracellular epitopes of this protein, specifically immunopreci pitated I-125-labeled Bolton-Hunter beta-subunit as well as [I-125]cha rybdotoxin 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 vi vo, the high conductance Ca2+-activated K+ channel exists as a multime r containing both alpha- and beta-subunits, and this cDNA represents t he first beta-subunit of a potassium channel cloned to date. Furthermo re, we demonstrate that the cloned protein is the subunit to which cha rybdotoxin is specifically and covalently incorporated when crosslinke d to the channel.