RECONSTITUTION OF EXPRESSED K-CA CHANNELS FROM XENOPUS-OOCYTES TO LIPID BILAYERS

Reconstitution of large conductance calcium-activated potassium (K-Ca) channels from native cell membranes into planar lipid bilayers provid es a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, K-Ca channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterolo gously expressed in Xenopus oocytes. In this report, we describe the r econstitution of cloned and expressed Slo K-Ca channels from Xenopus o ocyte membranes into lipid bilayers. The reconstituted channels demons trate functional properties characteristic of native K-Ca channels. Th ey possess a mean unitary conductance of approximate to 260 pS in symm etrical potassium (250 mM), and they are voltage- and calcium-sensitiv e. At 50 mu M Ca2+, their half-activation potential was near -20 mV; a nd their affinity for calcium is in the micromolar range. Reconstitute d Slo K-Ca channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethyl ammonium (K-D = 158 mu M, at 0 mV) and internal Ba2+ (K-D = 76 mu M, a t 40 mV). In addition, they were blocked by internally applied ''ball' ' inactivating peptide (K-D = 480 mu M, at 40 mV). These results demon strate that cloned K-Ca channels expressed in Xenopus oocytes can be r eadily incorporated into lipid bilayers where detailed mechanistic stu dies can be performed under controlled internal and external experimen tal conditions.