Purification and reconstitution of the high-conductance, calcium-activated potassium channel from tracheal smooth muscle.

Abstract

The high-conductance Ca(2+)-activated K+ (maxi-K) channel from bovine tracheal smooth muscle was purified to apparent homogeneity by a combination of conventional chromatographic techniques and sucrose density gradient centrifugation. Fractions with the highest specific activity for binding of monoiodotyrosine charybdotoxin, [125I]ChTX, were enriched approximately 2000-fold over the initial digitonin-solubilized material up to a specific activity of 1 nmol/mg protein. Silver staining after SDS-polyacrylamide gel electrophoresis of the fractions from the last step of the purification indicates that binding activity is correlated with a major component of the preparation that displays an apparent molecular weight of 62,000. Labeling the same preparation with 125I-Bolton-Hunter reagent reveals the existence of both 62 (alpha)- and 31 (beta)-kDa subunits, in an apparent stoichiometry of 1:1, comigrating with binding activity. The beta subunit is heavily glycosylated. Deglycosylation studies indicate that the beta subunit represents the protein to which [125I]ChTX is covalently incorporated in the presence of the bifunctional cross-linking reagent disuccinimidyl suberate. Binding of [125I]ChTX to the purified ChTX receptor displayed the same pharmacological profile that has been found previously for toxin binding to native membranes, including inhibition by iberiotoxin, limbatustoxin, tetraethylamonium, potassium, cesium, and barium. The purified preparation was reconstituted into liposomes which were then fused with artificial lipid bilayers. Single channels were readily observed with a conductance of 235 picosiemens in 150 mM KCl that displayed selectivity for potassium over chloride and that were blocked by ChTX. The open probability of these channels was increased by depolarizing membrane potentials and by raising the internal calcium concentration. These data suggest that the maxi-K channel purified from tracheal smooth muscle is composed of two subunits.