| Objective: large-conductance calcium-activated potassiumchannels (BKCa) from vascular smooth muscle cells directly take part in theregulation of vasodilatation, and play a role in the maintenance of bloodpressure. The studies of BKCa’s kinetics, regulate domain, target of drug action,meet many limitations using native cells. After reconstituting BKCachannelsinto planer lipid bilayers (PLBMs), all that studies could be easily done. Thepresent experiment is designed to establish a stable way of reconstitution ofPLBMs for ionic channel studies. By reconstituting cloned BKCachannels intoPLBMs and recording the single channel current, while with Coomassiebrilliant blue staining and Western blot analysis, the established method wasevaluated. Methods:(1) PLBMs experiments: Phosphatidylcholine(PC) or PCand CH were prepared firstly, and the PLBMs were reconstituted by paintingthese lipids on the aperture of the CUP; the composition of lipids and thesurrounding electrolyte were changed during the experiment to finally satisfythe stability of the PLBMs. In order to test the stability of the PLBMs, theliposome of amphotericin and α-toxin were added to the cis-compartment of theCUP. BKCaproliposomes were reconstituted into stable PLBMs and singlechannel currents were recorded.(2)Single channel experiment of BKCainPLBMs: The current of BKCachannels was recorded in symmetric high potassium solution by single channel recording technique, amplified by BC-535patch clamp amplifier,1KHz filtered, then imported into the computer, thedigital data was recorded by Clampex10.2software and analyzed by Clampfit10.1software.(3) Single BKCacurrent recording with Patch Clamp technique inHEK293cells, used as a control compare with the BKCain PLBMs: Chosestereo and in good state human embryonic kidney (HEK293) cells withtransfected BKCaα subunit for experiment. The current was recorded insymmetric high potassium solution. Single channel current was amplified byCEZ-2200patch clamp amplifier,1KHz filtered, then imported into thecomputer, the digital data was recorded by pClamp10.0software and analyzedby Clampfit10.1software.(4) Coomassie brilliant blue staining and Westernblot analysis to test the purity of BKCaproliposomes: Collected cell fragment ofHEK293cells transfected BKCaα subunit, which were treated byhyperisotonic solution, density gradient centrifugation in20%and38%sucrose/MOPS solution. Degenerated BKCaprotein samples were added toSDS-Polyacrylamide gel, then chose reasonable voltage to electrophoresis andelectroblotting. The membrane was incubated with primary polyclonalantibodies for the BKCaα subunit (1:500dilution) firstly, then the membranewas washed and incubated in goat-anti-rabbit IgG-HPR (secondary antibody,1:10,000dilution). The images were recorded by GEL-Doc gel imageformation systerm and analyzed by Quantity One software. Results:(1) PLBMsexperiment: the bilayers painted with PC alone could last3.5h, while the bilayers painted with PC and CH in different ratio could last2.5h. The resultsshow that when the liposome of amphotericin and α-toxin fused with PLBMs,the bilayers were stable.(2) The properties of single channel currents of BKCachannels in PLBMs were as follows:①The conductance of BKCachannelwas big: unitary conductance of BKCachannel was206.8±16.86pS (n=4).②The channel was voltage dependent. When the test voltage changed from-60mV to+60mV, the Amp and NPo increased significantly, To prolongated, Tcshortened.③The channel was calcium sensitive: when the calciumconcentration of CUP cis/trans bath changed from0mM to1μM and100μM,the NPo raised remarkably, showing a calcium activation.④The channel waspotassium selectivity: when the potassium concentration of CUP cis/trans bathwas140/140mM, the reversal potential of reconstituted BKCachannel wasapproximately0mV(n=4), while the potassium concentration of CUP cis/transbath was40/140mM, the reversal potential of reconstituted BKCachannel wasapproximately-30mV (n=2). The theoretical value according to the Nernstfunction of K+equilibrium potential (EK+) of140/140mM KCl was-32.6mV,and the EK+of140/140mM KCl was0mV.(3) The properties of single channelcurrents of BKCachannels in HEK293cells，Single channel currents of BKCawere recorded in inside-out patches.①The BKCachannel had big conductance:the conductance of BKCachannel was212.7±7.71pS（n=8）.②The channelwas voltage dependent. When the test voltage changed from0mV to+60mV,the Amp and NPo increased significantly, To prolongated, Tc shortened.③The channel was calcium sensitive: when the free calcium concentration of bathsolution changed from0M to10-7M、5×10-7M and10-6M, the NPo raisedremarkably, showing a calcium activation.(4) Results of Coomassie brilliantblue staining and Western blot analysis: The results obtained from the Westernblot and Coomassie brilliant blue staining indicated that the proliposomes ofour experiment contained BKCaα subunit, with it’s Molecular Weight of100kDa, and indicated that the proliposome had other interferential proteins.Conclusion:(1) PLBMs can be reconstituted successfully by painting method.(2) This method can be used to further research of BKCachannel onpharmacology and kinetics.(3) The reconstitued BKCachannel have characticssuch as voltage dependence, calcium sensitive, potassium selectivity.(4) theprepared proliposomes currently contained other proteins. |
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