Department of Physiology, University of Tübingen, Tübingen, Germany; and.
Institute of Pharmacy, Department of Pharmacology and Toxicology, University of Tübingen, Tübingen, Germany.
Am J Physiol Cell Physiol. 2014 Jun 1;306(11):C1041-9. doi: 10.1152/ajpcell.00209.2013. Epub 2014 Apr 2.
The iberiotoxin-sensitive large conductance voltage- and Ca(2+)-activated potassium (BK) channels (maxi-K(+)-channels) hyperpolarize the cell membrane thus supporting Ca(2+) entry through Ca(2+)-release activated Ca(2+) channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca(2+)-insensitive BK channels (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function (V617F)JAK2, or inactive (K882E)JAK2. K(+) conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K(+) current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K(+) current in BK(M513I+Δ899-903)-expressing oocytes was significantly increased following coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. Coexpression of the BK channel with (V617F)JAK2 but not (K882E)JAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and (V617F)JAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 μM) significantly decreased K(+) current. Inhibition of channel insertion by brefeldin A (5 μM) decreased the K(+) current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and (V617F)JAK2. The iberiotoxin (50 nM)-sensitive K(+) current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 μM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.
伊比利亚毒素敏感的大电导电压和 Ca(2+)-激活钾 (BK) 通道（最大 K(+)-通道）超极化细胞膜，从而支持通过 Ca(2+)-释放激活的 Ca(2+)通道进入 Ca(2+)。Janus 激酶-2 (JAK2) 已被确定为新型离子转运调节剂。为了探讨 JAK2 是否参与 BK 通道的调节，将编码 Ca(2+)-不敏感 BK 通道 (BK(M513I+Δ899-903)) 的 cRNA 与或不与编码野生型 JAK2、功能获得 (V617F)JAK2 或失活 (K882E)JAK2 的 cRNA 一起注入非洲爪蟾卵母细胞。通过双电极电压钳确定 K(+)电导，通过共聚焦显微镜确定 BK 通道蛋白丰度。在 A204 肺泡横纹肌肉瘤细胞中，利用全细胞膜片钳测定伊比利亚毒素敏感的 K(+)电流。进一步用 JAK2 和 BK 通道转录本转染 A204 细胞，并分别通过 RT-PCR 和 Western 印迹定量蛋白质丰度。结果，在共表达 JAK2 或 (V617F)JAK2 而不是 (K882E)JAK2 后，表达 BK(M513I+Δ899-903) 的卵母细胞中的 K(+)电流显著增加。BK 通道与 (V617F)JAK2 的共表达而不是 (K882E)JAK2 增强了卵母细胞膜上 BK 通道蛋白的丰度。将 BK 通道和 (V617F)JAK2 表达的卵母细胞暴露于 JAK2 抑制剂 AG490（40 μM）显著降低 K(+)电流。用布雷菲德菌素 A（5 μM）抑制通道插入将 K(+)电流降低到表达 BK 通道的卵母细胞和表达 BK 通道和 (V617F)JAK2 的卵母细胞中的相似程度。伊比利亚毒素（50 nM）预处理（40 μM，12 h）显著降低横纹肌肉瘤细胞中的伊比利亚毒素（50 nM）敏感的 K(+)电流。此外，在 A204 细胞中过表达 JAK2 显著增强了 BK 通道 mRNA 和蛋白丰度。总之，JAK2 通过增加细胞膜上的通道蛋白丰度来上调 BK 通道。
