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

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 (BK_Ca) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N₂-weathered mixture of phosphatidylcholine and cholesterol at 3:1 ratio. After successful incorporation of BK_Ca channel into PLBMs, single channel characteristics of BK_Ca were studied by patch clamp method. The results showed that i) the single channel conductance of BK_Ca was (206.8 ± 16.9) pS; ii) the activities of BK_Ca channel were voltage dependent; iii) in the bath solution without Ca²⁺, there was almost no BK_Ca channel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BK_Ca 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 BK_Ca 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 BK_Ca into PLBMs showed similar electrophysiological characteristics to natural BK_Ca channels, so the PLBMs with incorporated BK_Ca can be used in the studies of pharmacology and dynamics of BK_Ca channel.