Subunit composition of the high conductance calcium-activated potassium channel from smooth muscle, a representative of the mSlo and slowpoke family of potassium channels.

TLDR

The authors purified bovine tracheal and aortic smooth‑muscle maxi‑K channels using charybdotoxin binding and chromatography, then resolved the alpha subunit by SDS‑PAGE, digested it, isolated peptides by HPLC, and microsequenced them by Edman degradation. The purified channel is composed of a 62‑kDa alpha subunit and a 31‑kDa beta subunit; sequence analysis shows high homology to the mSlo maxi‑K channel, and an anti‑peptide antibody demonstrates that the alpha and beta subunits noncovalently associate, confirming the alpha subunit is a member of the mSlo family and that existing biochemical data on charybdotoxin receptors apply to native maxi‑K channels.

Abstract

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.