[Reconstitution of large conductance calcium-activated potassium channels into artificial planar lipid bilayers].

ABSTRACT

This study was aimed to establish a method to create a stable planar lipid bilayer membranes (PLBMs), in which large conductance calcium-activated potassium channels (BKCa) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N2-weathered mixture of phosphatidylcholine and cholesterol at 31 ratio. After successful incorporation of BKCa channel into PLBMs, single channel characteristics of BKCa were studied by patch clamp method. The results showed that i) the single channel conductance of BKCa was (206.8 ± 16.9) pS; ii) the activities of BKCa channel were voltage dependent; iii) in the bath solution without Ca2+, there was almost no BKCa channel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BKCa channels were activated in a Ca2+ concentration dependent manner; v) when [Ca2+] was increased from 1 µmol/L to 100 µmol/L, both the channel open probability and the average open time were increased, and the average close time was decreased from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms; vi) the reverse potential of the reconstituted BKCa was -30 mV when [K+] was at 40/140 mmol/L (Cis/Trans). These results suggest that the spreading method could serve as a new method for preparing PLBMs and the reconstituted BKCa into PLBMs showed similar electrophysiological characteristics to natural BKCa channels, so the PLBMs with incorporated BKCa can be used in the studies of pharmacology and dynamics of BKCa channel.