Identification of a Kv3.4 channel in corneal epithelial cells.
Identification of a Kv3.4 channel in corneal epithelial cells.
复制标题
角膜上皮细胞中 Kv3.4 通道的鉴定。
DOI：
10.1167/iovs.03-1056
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发表时间：
2004
影响因子：
4.4
通讯作者：
Lu,Luo
中科院分区：
文献类型：
--
作者：
Wang,Ling;Fyffe,RobertEW;Lu,Luo
目的.电压门控性K+通道维持角膜上皮细胞的水盐平衡和正常功能。为了确定他们的身份，KV通道类型培养的兔角膜上皮细胞和完整的大鼠角膜上皮。免疫组织化学和蛋白质印迹分析进行检测K+通道的大鼠和SV-40转化兔角膜上皮（RCE）细胞的膜和细胞裂解物，使用特异性抗体。全细胞膜片钳被用来表征RCE细胞中的K+电流的生物物理和药理学特性。用一组特异性抗K+通道抗体检测角膜上皮细胞K+通道类型的表达。蛋白质印迹分析，使用特异性抗K+通道抗体，包括抗Kv 1。1，-2.1，-3.1，-3.2，-3.4，-4.2，和-4.3，表明在角膜上皮细胞中Kv 3. 4通道在全细胞裂解物和细胞膜制备物中高度表达。反Kv 3 4通道抗体在RCE细胞和大鼠角膜上皮中均产生强烈的免疫反应性。荧光免疫染色和抗生物素蛋白-生物素-过氧化物酶复合物免疫染色证实了Kv 3的定位。RCE和大鼠角膜上皮细胞膜上的4个通道。电压去极化激活的K+电流在RCE细胞中被抑制的应用程序的4-氨基吡啶（4-AP，在微摩尔水平），α-树枝毒素在纳摩尔水平，或血液抑制物质-I在纳摩尔水平。大鼠和RCE细胞中电压门控的4-AP敏感性K+通道的生化和药理学特征类似于Kv 3的特征。4频道，肖氏亚家族的一员。该通道可能在维持角膜上皮正常功能中起重要作用。
purpose. Voltage-gated K+ channels maintain salt and water balance and normal function of corneal epithelial cells. To determine their identity, Kv channel types were sought in cultured rabbit corneal epithelial cells and in the intact rat corneal epithelium.methods. Immunohistochemistry and Western blot analysis were performed to detect K+ channels in the membrane and cell lysates of rat and SV-40–transformed rabbit corneal epithelial (RCE) cells, using specific antibodies. The whole-cell patch clamp was used to characterize the biophysical and pharmacologic properties of the K+ current in RCE cells.results. Expressions of K+ channel types in corneal epithelial cells were detected by using a panel of specific anti-K+ channel antibodies. Western blot analysis, using specific anti-K+ channel antibodies including anti-Kv1. 1,-2.1,-3.1,-3.2,-3.4,-4.2, and-4.3, demonstrated that in corneal epithelial cells Kv3. 4 channel was highly expressed in whole-cell lysates and in cell membrane preparations. The anti-Kv3. 4 channel antibody produced intense immunoreactivity in both RCE cells and rat corneal epithelium. Fluorescence immunostaining and avidin-biotin-peroxidase complex immunostaining confirmed localization of Kv3. 4 channels in the cell membrane of both RCE and rat corneal epithelial cells. Voltage depolarization-activated K+ currents in RCE cells were inhibited by applications of either 4-aminopyridine (4-AP, at micromolar levels), α-dendrotoxin at nanomolar levels, or blood-depressing substance-I at nanomolar levels.conclusions. Biochemical and pharmacological profiles of the voltage-gated, 4-AP–sensitive K+ channel in rat and RCE cells resemble characteristics of a Kv3. 4 channel, a member of the Shaw subfamily. This channel may play important roles in maintaining normal function of corneal epithelium.