PURIFICATION AND RECONSTITUTION OF THE HIGH-CONDUCTANCE, CALCIUM-ACTIVATED POTASSIUM CHANNEL FROM TRACHEAL SMOOTH-MUSCLE

The high-conductance Ca2+-aetivated K+ (maxi-K) channel from bovine tr acheal smooth muscle was purified to apparent homogeneity by a combina tion of conventional chromatographic techniques and sucrose density gr adient centrifugation. Fractions with the highest specific activity fo r binding of monoiodotyrosine charybdotoxin, [I-125]ChTX, were enriche d approximately 2000-fold over the initial digitonin-solubilized mater ial up to a specific activity of 1 nmol/mg protein. Silver staining af ter SDS-polyacrylamide gel electrophoresis of the fractions from the l ast step of the purification indicates that binding activity is correl ated with a major component of the preparation that displays an appare nt molecular weight of 62,000. Labeling the same preparation with, I-1 25-Bolton-Hunter reagent reveals the existence of both 62 (alpha)- and 31 (beta)-kDa subunits, in an apparent stoichiometry of 1:1, comigrat ing with binding activity. The beta subunit is heavily glycosylated. D eglycosylation studies indicate that the beta subunit represents the p rotein to which [I-125]ChTX is covalently incorporated in the presence of the bifunctional cross-linking reagent disuccinimidyl suberate. Bi nding of [I-125]ChTX to the purified ChTX receptor displayed the same pharmacological profile that has been found previously for toxin bindi ng to native membranes, including inhibition by iberiotoxin, limbatust oxin, tetraethylamonium, potassium, cesium, and barium. The purified p reparation 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 selecti vity for potassium over chloride and that were blocked by ChTX. The op en probability of these channels was increased by depolarizing membran e potentials and by raising the internal calcium concentration. These data suggest that the maxi-K channel purified from tracheal smooth mus cle is composed of two subunits.