Subunit composition of the high conductance calcium-activated potassium channel from smooth muscle, a representative of the mSlo and slowpoke family of potassium channels.
High conductance Ca(2+)-activated K+ (maxi-K) channels from bovine tracheal and aortic smooth muscle membranes have been purified employing monoiodotyrosine charybdotoxin binding as a marker for the channel and conventional chromatographic techniques. This K+ channel is composed of two subunits, alpha and beta, of 62 and 31 kDa, respectively. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electroeluted tracheal smooth muscle alpha-subunit was subjected to tryptic cleavage and a number of fragments were isolated by microbore C18 high performance liquid chromatography. Several of these peptides were microsequenced using Edman degradation techniques. Amino acid sequence information obtained from these fragments reveals the existence of very high sequence homology with the recently cloned mSlo maxi-K channel (Butler, A., Tsunoda, S., McCobb, D. P., Wei, A., and Salkoff, L. (1993) Science 261, 221-224). A specific anti-peptide antibody directed against the amino acid sequence of one of the fragments of the alpha-subunit is capable of specifically immunoprecipitating not only the denatured 125I-Bolton-Hunter-labeled alpha-subunit, but also, under nondenaturing conditions, the complex of alpha and beta subunits, demonstrating specific noncovalent association of both subunits. Thus, our results indicate that the alpha-subunit of the purified tracheal smooth muscle maxi-K channel is a member of the mSlo family of K+ channels and forms a noncovalent complex with a beta-subunit. It is concluded that the extensive biochemical information acquired to date on smooth muscle charybdotoxin receptors is pertinent to the structure of native maxi-K channels.
Knaus
H G
HG
Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065.
Garcia-Calvo
M
M
Kaczorowski
G J
GJ
Garcia
M L
ML
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000737:chemistry; D002417:Cattle; D018999:Charybdotoxin; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000737:chemistry*; D009131:Muscle, Smooth, Vascular / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry* / Q000145:classification; D012604:Scorpion Venoms / Q000378:metabolism
D046911:Macromolecular Substances; D015221:Potassium Channels; D012604:Scorpion Venoms; D018999:Charybdotoxin