Author + information
- Cheng Jun,
- Xiaorong Zeng,
- Xiaoqiu Tan,
- Jing Wen,
- Pengyun Li,
- Wen Zhou and
- Yan Yang
large-conductance calcium-activated potassium channels (BKCa) from vascular smooth muscle cells directly take part in the regulation of vasodilatation, and play a role in the maintenance of blood pressure. The studies of BKCa's kinetics, regulate domain, target of drug action, meet many limitations using native cells. After reconstituting BKCa channels into planer lipid bilayers (PLBMs), all that studies could be easily done. The present experiment is designed to establish a stable way of reconstitution of PLBMs for ionic channel studies. By reconstituting cloned BKCa channels into PLBMs and recording the single channel current, while with Coomassie brilliant blue staining and Western blot analysis, the established method was evaluated.
PLBMs experiments: Phosphatidylcholine(PC) or PC and Cholesteryl (CH) were prepared firstly, and the PLBMs were reconstituted by painting these lipids on the aperture of the CUP; the composition of lipids and the surrounding electrolyte were changed during the experiment to finally satisfy the stability of the PLBMs. In order to test the stability of the PLBMs, the liposome of amphotericin and α-toxin were added to the cis-compartment of the CUP. BKCa proliposomes were reconstituted into stable PLBMs and single channel currents were recorded. Single channel experiment of BKCa in PLBMs. Single BKCa current recording with Patch Clamp technique in HEK 293 cells, used as a control compare with the BKCa in PLBMs.
Preparation of different concentrations of lecithin PC PLBMs stable 3.5h, and with different proportion of PC and CH were prepared by PLBMs can stable 2.5h reconstruction to the basic properties of the planar bilayer lipid membrane BKCachannel single channel.
smear method can be used for preparation of planar bilayer lipid membrane; planar bilayer lipid membrane technology can be used for BKCa channel pharmacology and kinetics study; reconstruction of BKCa with conductance, voltage dependent Ca2+ sensitivity and K+ selective.