Mederos y Schnitzler M, Derst C, Daut J, Preisig-Müller R
Institut fur Normale und Pathologische Physiologie, Universitat Marburg, Deutschhausstrasse 2, D-35037 Marburg, Germany.
J Physiol. 2000 Jun 1;525 Pt 2(Pt 2):307-17. doi: 10.1111/j.1469-7793.2000.t01-1-00307.x.
The full-length cDNAs of two different alpha-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different beta-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3-15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 +/- 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 microM 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. Rilmakalim (1 microM), diazoxide (300 microM) and metabolic inhibition increased the slope conductance measured at -55 mV by a factor of 9.0 (+/-1.8), 2.5 (+/-0.2) and 3.9 (+/-1.7), respectively. The effects were reversed by glibenclamide (1 microM). Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall.
通过筛选豚鼠心脏心室的cDNA文库，获得了豚鼠（gp）ATP敏感性钾（KATP）通道的两种不同α亚基（Kir6.1和Kir6.2）的全长cDNA以及三种不同β亚基（SUR1、SUR2A和SUR2B）的部分cDNA。使用基因特异性内含子跨越引物进行细胞特异性逆转录酶PCR表明，gpKir6.1、gpKir6.2和gpSUR2B在纯化的毛细血管内皮细胞组分中表达。在心肌细胞中，检测到了gpKir6.1、gpKir6.2、gpSUR1和gpSUR2A。在由3 - 15个内皮细胞组成的分离毛细血管片段中进行了膜片钳测量。在全细胞模式下测量的膜电容为19.9±1.0 pF，且与毛细血管片段的长度无关，这表明在我们的实验条件下内皮细胞没有电耦合。采用穿孔膜片技术测量毛细血管内皮细胞的稳态电流 - 电压关系。应用钾通道开放剂（瑞马卡林或二氮嗪）或代谢抑制（250 μM 2,4 - 二硝基苯酚加10 mM脱氧葡萄糖）诱导出一种在计算的钾平衡电位附近反转的电流。瑞马卡林（1 μM）、二氮嗪（300 μM）和代谢抑制分别使在 - 55 mV测量的斜率电导增加了9.0（±1.8）倍、2.5（±0.2）倍和3.9（±1.7）倍。这些效应被格列本脲（1 μM）逆转。我们的结果表明，豚鼠心脏的毛细血管内皮细胞表达由SUR2B和Kir6.1和/或Kir6.2亚基组成的KATP通道。KATP通道开放引起的超极化可能导致游离胞质Ca2+增加，从而调节一氧化氮的合成和毛细血管壁的通透性。
