title: Primary sequence and immunological characterization of beta-subunit of high conductance Ca(2+)-activated K+ channel from smooth muscle.
abstract: 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(2+)-activated K+ 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 125I-labeled Bolton-Hunter beta-subunit as well as [125I]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(2+)-activated K+ 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.
authors.affiliation: Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065.
authors.forename: H G
authors.initials: HG
authors.lastname: Knaus
authors.forename: K
authors.initials: K
authors.lastname: Folander
authors.forename: M
authors.initials: M
authors.lastname: Garcia-Calvo
authors.forename: M L
authors.initials: ML
authors.lastname: Garcia
authors.forename: G J
authors.initials: GJ
authors.lastname: Kaczorowski
authors.forename: M
authors.initials: M
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Swanson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D015870:Gene Expression; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry*; D015221:Potassium Channels / Q000737:chemistry* / Q000276:immunology*; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; D009124:Muscle Proteins; D015221:Potassium Channels; D012333:RNA, Messenger; C087375:charybdotoxin receptor