Functional reconstitution of the large-conductance, calcium-activated potassium channel purified from bovine aortic smooth muscle.
The charybdotoxin (ChTX) receptor has been purified from bovine aortic smooth muscle using conventional chromatographic techniques and sucrose gradient centrifugation. Fractions from the final sucrose gradient purification were enriched in specific binding of monoiodinated ChTX (125i-ChTX) approximately 2000-fold over native sarcolemmal membranes. The ChTX binding activity correlated with the presence of two polypeptides of 65 (alpha) and 31 (beta) kDa. Using the cross-linking reagent, disuccinimidyl suberate, 125I-ChTX was specifically incorporated into a polypeptide of approximately 31 kDa. Cross-linking and binding of 125I-ChTX to the purified ChTX receptor was inhibited by ChTX, iberiotoxin (IbTX), and tetraethylammonium (TEA). Liposomes containing the purified ChTX receptor were incorporated into planar lipid bilayers. In symmetric 150 mM KC1, the channels observed were > 20-fold more selective for potassium over sodium and exhibited a large, single-channel conductance of 323 +/- 2.5 pS in charged lipids and 249 +/- 7 pS in neutral lipids. Depolarizing membrane potentials increased the open probability of the purified channels e-fold per 11.5 +/- 0.3 mV, while intracellular calcium increased the open probability according to a third power (2.9 +/- 0.2) relationship. Mean channel closed durations decreased while open times slightly increased as membrane potential and calcium concentration were elevated. The distributions of open and closed durations were well described by the sums of three and five to six exponential components, respectively. Purified maxi-K channels were blocked with micromolar affinity by external TEA and with nanomolar affinity by extracellular IbTX and ChTX. Kinetics of ChTX block of the purified channel revealed an equilibrium dissociation constant for toxin block 4.6 +/- 0.7 nM under conditions of physiological ionic strength. The purified maxi-K channel displays many of the biophysical and pharmacological properties of maxi-K channels derived from native tissue.
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D018999:Charybdotoxin / Q000378:metabolism / Q000494:pharmacology; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D015640:Ion Channel Gating; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000302:isolation & purification / Q000378:metabolism*; D013388:Succinimides; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D013388:Succinimides; D013757:Tetraethylammonium Compounds; C087375:charybdotoxin receptor; D018999:Charybdotoxin; D019789:Tetraethylammonium; C064719:iberiotoxin; D002118:Calcium; C019358:disuccinimidyl suberate