Effects of isoflurane and ethanol on large conductance Ca2+ -activated K+ channels

Journal of Medical Colleges of PLA 2004; 19(3)Effects of isoflurane and ethanol on large conductance Ca2+-activated KchanneeIsWANG Ying-wei(王英伟),Ⅺ IONG Yuan-chang(熊源长), DENG Xiao-ming(邓小明)DetSecond Military Medical University, Shanghai 200433, ChinaAbstract Objective To study the effect of isoflurane and ethanol on large conductance Ca-activated K channe(BK channels). Methods: The cRNA of mslol encoding BK channels was injected into Xenopuscubated in ND96(96 mmol/L NaCl, 2.0 mmol/L KCl, 1.8 mmol/L CaCI,, 1. 0 mmol/L MgCl,no∥/ L HEPES,pH 7.4)at 4C. Patch clamp recording(outside-out )were performed after 2-3 d. Isoflurane was administrated by the vaporizer driven by air, ethanol was applied by a closed, manual-controlled administration system. Different test potentials from0 to 10 mv were given to observe changes of currents. Results: 0. 7 mmol/L and 1. 2 mmol/L of isoflurane could inhibit BKcurrents obviously at different command potentials, but 50 mmol/L, 100 mmol/L, or 200 mmol/L of ethanol had no any ef.fect on BK currents. Conclusion: Clinical concentration of isoflurane can distinctly inhibit isolating BK currentsKey words isoflurane; ethanol; patch clamp: large conductance Ca-activated K channelsLarge conductance Ca-activated K channel( BK Macroscopic currents were induced by Ag/AgCl pipetlechannel)is widely located in neurons, vascular smooth which were maximum frequency at 10 kHz with analog fil-muscles, and endothelial cells. It is believed that BK tering during acquisition, and recorded in computer bychannel plays an important role in excitability of cells. Digidata 1200 AD/DA transfer panel and pclamp 8. 0 softWhen BK channels were activated, intracellular K* efflux ware. Extracellular solution included 140 mmol/L potassilead to hyperpolarization. It has been indicated that vol- um methanesulfonate, 20 mmol/L KOH, 10 mmol/Latile agents might contribute to general anesthesia by acting HEPES, and 2 mmol/L MgCI, pH7.0. The pipette soluon potassium channels. Therefore, we designed this exper- tion contained ND96 and 0. 5 umol/L CaCI. Pclamp 8.0iment to study the effect of clinical relevant concentration of for Windows was used to generate voltage commands and tosoflurane on bK channeldigitize currents. Holding potential was 0 mV, commanMATERIALS AND METHODSpotential was from 170 to- 140 mV with 10 mV incre-mentsExpression in Xenopus oocytes All Xenopus(pro-Method of administration The isoflurane vaporizerof Washington university in St. Lou- was checked and adjusted before experiments. The inputis)were anesthetized with dry CO2. Oocytes were taken out terminal of vaporizer was connected to air in the wall, theand maintained in ND96 solution( NaCl 96.0 mmol/L, output teminal was connected to recording chamber whichKCI 2.0 mmol/L, CaCI2 1. 8 mmol/L, MgCI, 1. 8 mmol/ L, contained oocytes in a glass tube. When isoflurane was ap-and HEPES 5.0 mmol/L, pH 7. 4)supplemented with plied to patches, the extracellular solutions were sealedpenicillin(100 U/ml), streptomycin(100 mg/ml), and tightly and measured by gas chromatography after recordgentamycin(50 mg/ml), and reserved in refrigerator at 17 ing. Ethanol was diluted into 50, 100, and 200 mmol/L byC. The cRNA(10-50 nl)of mslol which would express extracellular solution, and applied to patches by gravityBK channels was injected into cytoplasm of Xenopus oo-Statistical analysisdata were expressed withcytes by microinjection. The oocytes were incubatedMean SD, and analyzed by Clampfit 8.0 software. PeakND96, and used for electrophysiological experiments 2-3 current中国煤化工at different commandd after injectionpotentiCNMHGElectrophysiological recording For this experiment, REsULTscurrents were recorded in the outside-out patch modeThe typical BK currents were obtained from outside-*ComespondingauthorDFNGXiao-ming.E-mail:Deng-x@yahoo.comoutpatchbydifferentcommandpotentials,whichwascon-182Journal of Medical Colleges of PLA 2004; 19(3)sistent with our previouivated potassium channels classified by their unitpatches were obtained in this experiment(12 patches on conducatnces: large(BK), intermediate( IK), and smallisoflurane, 20 patches on ethanol). As shown in Fig 1,( SK)conductance. BK channel is gated by Ca and volt-the inhibitory effect of isoflurane on BK channel was volt- age, while IK and SK channels are solely gated by Caage-dependent. Clinical relevant conceIt has been demonstrated,that volatile anestheticshad little effects on BK currents when command potential such as halothane, enflurane, isoflurane, and sevoflurane,was between -10 to-140 mv. But the inhibitory effect suppress the activity of IK channel, but no effect on SKwas enhanced dramatically when command potential was channel. There are less reports on BK channels associatedmore than 10 mV. The inhibition was impaired again when with volatile agents. It has been indicated that at a freecommand potential was more than 30 mV. The maximum cytoplasmic calcium concentration of I pmmol/L, the clininhibition of 0.7 and 1.2 mmol/L isoflurane on BK currents cal relevant dose of 0. 5 mmol/L halothane reduced thwas 80% and 90%, respectively, when command poten- open probability of BK channel without altering the single-tial was 30 mV. There were no effects of 50, 100, and 200 channel conductance. Similarly, an inside-out patchmmol/L ethanol on BK channels( Fig 2)recording indicated that clinical relevant concentration ofketamine selectively blocked BK channel via the competition of Ca+ and ketamine. This inhibition was due to a-e-1. 2 mmol/L Iso-o-0. 7 mmol/L IsOdecrease in mean closed time but no decrease in singli政政channel current amplitude. The method of administrationis not a physiological way based on the model of inside-outpatch recording. Therefore, we investigated the effect ofIsoflurane onby the model of outside-outpatch. Our results indicated that clinical relevant concen-tration of isoflurane inhibited BK channel in a reversibleoltage-dependent manner. For the intracellular K efflux0200ommand polential (U/mvof BK channel, the inhibitory effect of isoflurane on BKchannel might increase the excitability of cellsFig 1 The inhibitory effect of isoflurane( ISo)on BKRecently, Gruss et al 6 investigated the effect of eth-anol on BK channel in rat dorsal root ganglion by outside-out patch; it was found that clinical relevant concentrationof ethanol(40-80 mmol)increased the open probability的略of channel by 2-6 times, and the excitability of BK channel might contribute to clinically well-kncduced analgesia and paresthesia. However, our study indi100 mmol/L ethanol200 mmol/L ethanolated that 50, 100, and 200 mmol/L ethanol had no effect0.4on BK channel encoded with mslol. The contradictive reults may be interpreted by the difference of gene encodingacid resulted in unlikepharmacology between rat and mouseFig 2 No effect of ethanol on BK(mslol )channelvant concentration of isoflurane is likely to block BK chaYH中国煤化工DISCUSSIONCNMHGCa*-activated potassium channels regulate a wide va- 1 Calderone V. Large-conductance, Ca-activated K "channels: functionriety of cellular functions by coupling intracellular Charmacology and drugs[J]. Curr Med Chen, 2002; 9(14):1385concentration to membrane potential. There are three major(To be continued on page 186)186Journal of Medical Colleges of PLA 2004; 19(3)( Panis),1996;44(10):867dence for involvement of both cell mediated and humoral immunity in[3] Al Abadie MS, Senior H], Bleehen SS et al. Neuropeptide and neuronalgeneralized vitiligo[J]. Pigment Cell Res, 1994; 7:Imarker studies in vitiligo J]. Br J Dermatod, 1994:131: 160[11] Tu CX, Fu HW, Lin XR. 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Proc Natl Aced Sci USA, 1995:95Endocrinology,2002;143(5):1686( Received2003-1203, revised200403-10)[10] Abdel Naser MB, Kruger Krasagakes S, Krasagakis K et al.Further evEditor XU Fu-mingContinued from page 182)5 Denson DD, Eaton DC. Ketamine inhibition of large conductance Cr22 Wang YW, Ding JP, Xia XM et al. Consequences of the stoichiometry oftivated K+ channels is modulated by intracellular Ca*[J]. Am J PhysiolSIol alpha and auxiliary beta subunits on functional properties of large-con-94;267(5P1):C1452uctance Ca2+activated K+ channels [J]. J Neurosci, 2002: 22(5): 6 Gruss M, Henrich M, Konig P et al. Ethanol reduces excitability in a sub-group of primary sensory neurons by activation of BK( Ca)channels[J]3 Namba T. Ishii TM, Ikeda M. Inhibition of the human intermediate con-Fur neurosci,2001;l4(8);1246ductance Ca2-activated K' channel, hIKI, by volatile anesthetics[J]. 7 Xia XM, Ding JP, Lingle CJ. Molecular basis for the inactivation of Ca?'Eur j pharmaco,2000;28:395(2):95and voltage dependent BK channels in adrenal chromaffin cells and rat inHong Y, Puil E, Mathers DA. Eect of halothane on large-conductancesulinoma tumor cells[J]. J Neurosci, 1999; 19(13):5255ealcium-dependent potassium channels in cerebrovascular smooth muscleReceived 2003-09-01, revised 2004-01-12)cells of the rat[ J]. Anesthesiology, 1994: 81(3):649(Editor CAO Jing, I Dan-yang)中国煤化工CNMHG