McGahon Mary K, Dawicki Jennine M, Arora Aruna, Simpson D A, Gardiner T A, Stitt A W, Scholfield C Norman, McGeown J Graham, Curtis Tim M
Centre of Vision Sciences, The Queen's University of Belfast, Institute of Clinical Sciences, The Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA. UK.
Am J Physiol Heart Circ Physiol. 2007 Feb;292(2):H1001-8. doi: 10.1152/ajpheart.01003.2006. Epub 2006 Oct 13.
Little is known about the molecular characteristics of the voltage-activated K(+) (K(v)) channels that underlie the A-type K(+) current in vascular smooth muscle cells of the systemic circulation. We investigated the molecular identity of the A-type K(+) current in retinal arteriolar myocytes using patch-clamp techniques, RT-PCR, immunohistochemistry, and neutralizing antibody studies. The A-type K(+) current was resistant to the actions of specific inhibitors for K(v)3 and K(v)4 channels but was blocked by the K(v)1 antagonist correolide. No effects were observed with pharmacological agents against K(v)1.1/2/3/6 and 7 channels, but the current was partially blocked by riluzole, a K(v)1.4 and K(v)1.5 inhibitor. The current was not altered by the removal of extracellular K(+) but was abolished by flecainide, indicative of K(v)1.5 rather than K(v)1.4 channels. Transcripts encoding K(v)1.5 and not K(v)1.4 were identified in freshly isolated retinal arterioles. Immunofluorescence labeling confirmed a lack of K(v)1.4 expression and revealed K(v)1.5 to be localized to the plasma membrane of the arteriolar smooth muscle cells. Anti-K(v)1.5 antibody applied intracellularly inhibited the A-type K(+) current, whereas anti-K(v)1.4 antibody had no effect. Co-expression of K(v)1.5 with K(v)beta1 or K(v)beta3 accessory subunits is known to transform K(v)1.5 currents from delayed rectifers into A-type currents. K(v)beta1 mRNA expression was detected in retinal arterioles, but K(v)beta3 was not observed. K(v)beta1 immunofluorescence was detected on the plasma membrane of retinal arteriolar myocytes. The findings of this study suggest that K(v)1.5, most likely co-assembled with K(v)beta1 subunits, comprises a major component underlying the A-type K(+) current in retinal arteriolar smooth muscle cells.
关于体循环血管平滑肌细胞中构成A 型钾电流基础的电压门控钾(K(v))通道的分子特征,人们了解甚少。我们使用膜片钳技术、逆转录聚合酶链反应(RT-PCR)、免疫组织化学和中和抗体研究,调查了视网膜小动脉肌细胞中A 型钾电流的分子身份。A 型钾电流对K(v)3 和K(v)4 通道的特异性抑制剂的作用具有抗性,但被K(v)1 拮抗剂correolide 阻断。针对K(v)1.1/2/3/6 和7 通道的药物未观察到任何作用,但该电流被K(v)1.4 和K(v)1.5 抑制剂利鲁唑部分阻断。去除细胞外钾(K(+))不会改变该电流,但氟卡尼可将其消除,这表明是K(v)1.5 而非K(v)1.4 通道。在新鲜分离的视网膜小动脉中鉴定出编码K(v)1.5 而非K(v)1.4 的转录本。免疫荧光标记证实缺乏K(v)1.4 表达,并显示K(v)1.5 定位于小动脉平滑肌细胞的质膜。细胞内应用抗K(v)1.5 抗体可抑制A 型钾电流,而抗K(v)1.4 抗体则无作用。已知K(v)1.5 与K(v)β1 或K(v)β3 辅助亚基共表达可将K(v)1.5 电流从延迟整流电流转变为A 型电流。在视网膜小动脉中检测到K(v)β1 mRNA 表达,但未观察到K(v)β3。在视网膜小动脉肌细胞质膜上检测到K(v)β1 免疫荧光。本研究结果表明,K(v)1.5 很可能与K(v)β1 亚基共同组装,是视网膜小动脉平滑肌细胞中A 型钾电流的主要组成成分。