Primary sequence and immunological characterization of beta-subunit of high conductance Ca(2+)-activated K+ 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(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.
Knaus
H G
HG
Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065.
Folander
K
K
Garcia-Calvo
M
M
Garcia
M L
ML
Kaczorowski
G J
GJ
Smith
M
M
Swanson
R
R
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
D008562:Membrane Glycoproteins; D009124:Muscle Proteins; D015221:Potassium Channels; D012333:RNA, Messenger; C087375:charybdotoxin receptor