FUNCTIONAL RECONSTITUTION OF THE LARGE-CONDUCTANCE, CALCIUM-ACTIVATEDPOTASSIUM CHANNEL PURIFIED FROM BOVINE AORTIC SMOOTH-MUSCLE

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.