Reconstitution of large conductance calcium-activated potassium channels into artificial planar lipid bilayers / 生理学报

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 (BK) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N-weathered mixture of phosphatidylcholine and cholesterol at 31 ratio. After successful incorporation of BKchannel into PLBMs, single channel characteristics of BKwere studied by patch clamp method. The results showed that i) the single channel conductance of BKwas (206.8 ± 16.9) pS; ii) the activities of BKchannel were voltage dependent; iii) in the bath solution without Ca, there was almost no BKchannel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BKchannels were activated in a Caconcentration dependent manner; v) when [Ca] 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 BKwas -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 BKinto PLBMs showed similar electrophysiological characteristics to natural BKchannels, so the PLBMs with incorporated BKcan be used in the studies of pharmacology and dynamics of BKchannel.