M. Garciacalvo et al., PURIFICATION AND RECONSTITUTION OF THE HIGH-CONDUCTANCE, CALCIUM-ACTIVATED POTASSIUM CHANNEL FROM TRACHEAL SMOOTH-MUSCLE, The Journal of biological chemistry, 269(1), 1994, pp. 676-682
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.