Conforti L, Bodi I, Nisbet J W, Millhorn D E
Department of Molecular Physiology, Institute of Molecular Pharmacology and Biophysics, College of Medicine, University of Cincinnati, Ohio, USA.
J Physiol. 2000 May 1;524 Pt 3(Pt 3):783-93. doi: 10.1111/j.1469-7793.2000.00783.x.
One of the early events in O2 chemoreception is inhibition of O2-sensitive K+ (KO2) channels. Characterization of the molecular composition of the native KO2 channels in chemosensitive cells is important to understand the mechanism(s) that couple O2 to the KO2 channels. The rat phaeochromocytoma PC12 clonal cell line expresses an O2-sensitive voltage-dependent K+ channel similar to that recorded in other chemosensitive cells. Here we examine the possibility that the Kv1.2 alpha-subunit comprises the KO2 channel in PC12 cells. Whole-cell voltage-clamp experiments showed that the KO2 current in PC12 cells is inhibited by charybdotoxin, a blocker of Kv1.2 channels. PC12 cells express the Kv1.2 alpha-subunit of K+ channels: Western blot analysis with affinity-purified anti-Kv1.2 antibody revealed a band at approximately 80 kDa. Specificity of this antibody was established in Western blot and immunohystochemical studies. Anti-Kv1.2 antibody selectively blocked Kv1.2 current expressed in the Xenopus oocyte, but had no effect on Kv2.1 current. Anti-Kv1.2 antibody dialysed through the patch pipette completely blocked the KO2 current, while the anti-Kv2.1 and irrelevant antibodies had no effect. The O2 sensitivity of recombinant Kv1.2 and Kv2.1 channels was studied in Xenopus oocytes. Hypoxia inhibited the Kv1.2 current only. These findings show that the KO2 channel in PC12 cells belongs to the Kv1 subfamily of K+ channels and that the Kv1.2 alpha-subunit is important in conferring O2 sensitivity to this channel.
氧化学感受的早期事件之一是对氧敏感的钾离子（KO2）通道受到抑制。确定化学感受细胞中天然KO2通道的分子组成，对于理解将氧与KO2通道偶联的机制很重要。大鼠嗜铬细胞瘤PC12克隆细胞系表达一种与其他化学感受细胞中记录到的类似的氧敏感电压依赖性钾离子通道。在此，我们研究了Kv1.2α亚基构成PC12细胞中KO2通道的可能性。全细胞膜片钳实验表明，PC12细胞中的KO2电流受到Kv1.2通道阻滞剂蝎毒素的抑制。PC12细胞表达钾离子通道的Kv1.2α亚基：用亲和纯化的抗Kv1.2抗体进行的蛋白质印迹分析显示在约80 kDa处有一条带。该抗体的特异性在蛋白质印迹和免疫组织化学研究中得到证实。抗Kv1.2抗体选择性地阻断非洲爪蟾卵母细胞中表达的Kv1.2电流，但对Kv2.1电流无影响。通过膜片吸管透析的抗Kv1.2抗体完全阻断了KO2电流，而抗Kv2.1抗体和无关抗体则无作用。在非洲爪蟾卵母细胞中研究了重组Kv1.2和Kv2.1通道的氧敏感性。低氧仅抑制Kv1.2电流。这些发现表明，PC12细胞中的KO2通道属于钾离子通道的Kv1亚家族，且Kv1.2α亚基对于赋予该通道氧敏感性很重要。
