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.
Highlights
From the Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, biochemical means
After sodium dodecyl sulfate-polyacrylamidegel electrophoresis, the electrocharybdotoxin (12sI-ChTX), high affinityreceptor siteshave been identified in bovine aortic and tracheal smooth muscle sarcolemma andshown to be associatedwith maxi-K channels
The ChTX receptor has been purified to homogeneity from bovine tracheal smooth musclemembranes (8)and shown eluted tracheal smooth musclea-subunit was subjected to consist of two noncovalently associated subunits with peak to tryptic cleavage and a number of fragments were iso- mobilities on SDS-PAGE corresponding to M, values of 62,000 lated by microbore Cle high performanceliquid chroma- and 31,000
Summary
Electroelution and nyptic Fragmentation of the Bovine lkacheal Maxi-K Channel a-Subunit-Fractions from the final sucrose density gradient centrifugation step containing -70 pmol of 'T-ChTX binding activity were dialyzed overnight against 5 rnTris-HC1, pH 7.4, 0.05% Maxi-KChannel a-Subunit Is a Representative of mSlo Family of X+ Channels-% obtain internal amino acid sequence information from the a-subunit of the bovine tracheal smooth muscle maxi-K channel, substantial quantities of this protein were purified by a combination of classical chromatographic steps and sucrose density gradient centrifugation.
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