埃他卡林对K_(ATP)通道亚型选择性作用的研究

Selective actions of iptakalim on the subtypes of K_(ATP) channels

目的建立特异性表达以K ir6.x和SUR亚单位组成的不同KATP通道亚型的细胞模型,研究KATP通道开放剂埃他卡林对KATP通道亚型的选择性作用特点。方法利用脂质体转染的方法将编码KATP通道亚单位K ir6.x-pcDNA3、SUR-pcDNA3基因和编码绿色荧光蛋白pEGFP-N1基因共同转染到HEK-293细胞中,免疫组化法鉴定细胞上K ir6.x和SUR蛋白的表达,并采用膜片钳全细胞记录技术,以KATP通道特异性激动剂吡那地尔和二氮嗪为对照,分析埃他卡林对特定刺激条件下对K ir6.x和SUR共同转染的细胞上诱发的钾电流的影响,并观察KATP通道特异性拮抗剂格列苯脲的阻断作用。结果在共同转入KATP通道两亚单位基因的HEK-293细胞上,有K ir6.x和SUR蛋白的表达。在共同表达K ir6.1/SUR2B的细胞上,埃他卡林(100μmol.L-1)、吡那地尔(100μmol.L-1)及二氮嗪(200μmol.L-1)均具有增强诱发电流的效应,给药前后-100 mV时电流值由(-88.0±30.8)pA分别增至(-2042.6±127.3)pA,(-1431.9±142.4)pA及(-1104.7±228.9)pA,三者的作用均能被格列苯脲所阻断。相同条件下,在共同表达K ir6.2/SUR2A的细胞上,埃他卡林和吡那地尔能增强诱发电流,此作用对格列苯脲敏感;而二氮嗪对该电流无影响。相反,在共同表达K ir6.2/SUR1的细胞上,二氮嗪能增强诱发电流,此作用对格列苯脲敏感;而埃他卡林和吡那地尔对该电流无影响。结论不同结构钾通道开放剂对不同亚型KATP通道作用不同,埃他卡林与吡那地尔作用相似,能选择性作用于K ir6.1/SUR2B和K ir6.2/SUR2A型KATP通道,对K ir6.2/SUR1型无影响。

Aim To establish a cell model heterologously expressing Kit6. x and SUR subunits of KATP channels and to study the selectivity of iptakalim on different subtypes of KATP channels. Methods Cell were transfected with the pcDNA vector containing the coding sequenced of Kir6. x and SUR using liposome and the pEGFP-N1 vector encoding for green fluorescent protein was added for easy identification of transfected cells. Using immunocytochemical technique, we detected the expression of Kir6. x and SUR protein in transfected ceils. The electrophysiological experiment was performed after transfection 48-72 h. In whole cell configuration, the effects of KATP channel openers iptakalim, pinacidil and diazoxide on the clone currents in transfected HEK-293 cells and the antagonism of KATP channel inhibitor glibenclamide were evaluated. Results Immunocytochemical study identified the expression of Kit6. x and SUR protein in transfected cells. The electrophysiological experiment showed that in cells with recombinant expression of the Kir6. 1/SUR2B channel complex, iptakalim （100 μm）, pinacidil（ 100 μm） and diazoxide（200μm） significantly increased the clone current from （ -88.0 ± 30. 8） pA to （ -2042.6 ±127.3） pA, （ - 1431.9 ± 142. 4） pA and （ - 1104. 7 ± 228.9） pA,respectively, at -100 mV, and the actions were inhibited by glibenclamide （10 μm ）. In cells expressed with Kir6. 2/SUR2A channel, both iptakalim and pinacidil activated the currents, which was sensitive to glibenclamide. While diazoxide had no effects on the clone currents. In contrast, in cells with Kir6. 2/ SUR1 channel, diazoxide increased the activity of recombinant KATP channels, while iptakalim and pinacidil had little effects. Conclusion From these observations, the effects of KATP channels openers with different chemical structures on the subtypes of KATP channels were diverse. Iptakalim showed selective action on Kir6. 1/SUR2B and Kir6. 2/SUR2A, but not Kir6.2/ SUR1 KATP channels.