Km. Giangiacomo et al., FUNCTIONAL RECONSTITUTION OF THE LARGE-CONDUCTANCE, CALCIUM-ACTIVATEDPOTASSIUM CHANNEL PURIFIED FROM BOVINE AORTIC SMOOTH-MUSCLE, Biochemistry, 34(48), 1995, pp. 15849-15862
The charybdotoxin (ChTX) receptor has been purified from bovine aortic
smooth muscle using conventional chromatographic techniques and sucro
se gradient centrifugation. Fractions from the final sucrose gradient
purification were enriched in specific binding of monoiodinated ChTX (
I-125-ChTX) approximately 2000-fold over native sarcolemmal membranes,
The ChTX binding activity correlated with the presence of two polypep
tides of 65 (alpha) and 31 (beta) kDa. Using the cross-linking reagent
, disuccinimidyl suberate, I-125-ChTX was specifically incorporated in
to a polypeptide of approximately 31 kDa, Cross-linking and binding of
I-125-ChTX to the purified ChTX receptor was inhibited by ChTX, iberi
otoxin (IbTX), and tetraethylammonium (TEA). Liposomes containing the
purified ChTX receptor were incorporated into planar lipid bilayers. I
n symmetric 150 mM KCl, the channels observed were >20-fold more selec
tive for potassium over sodium and exhibited a large, single-channel c
onductance of 323 +/- 2.5 pS in charged lipids and 249 +/- 7 pS in neu
tral lipids, Depolarizing membrane potentials increased the open proba
bility of the purified channels e-fold per 11.5 +/- 0.3 mV, while intr
acellular calcium increased the open probability according to a third
power (2.9 +/- 0.2) relationship. Mean channel closed durations decrea
sed while mean open times slightly increased as membrane potential and
calcium concentration were elevated, The distributions of open and cl
osed durations were well described by the sums of three and five to si
x exponential components, respectively. Purified maxi-K channels were
blocked with micromolar affinity by external TEA and with nanomolar af
finity by extracellular IbTX and ChTX. Kinetics of ChTX block of the p
urified channel revealed an equilibrium dissociation constant for toxi
n block of 4.6 +/- 0.7 nM under conditions of physiological ionic stre
ngth. The purified maxi-K channel displays many of the biophysical and
pharmacological properties of maxi-K channels derived from native tis
sue.