Serine protease activation of near-silent epithelial Na+ channels
Serine protease activation of near-silent epithelial Na+ channels
复制标题
DOI：
10.1152/ajpcell.00342.2003
复制
发表时间：
2004-01-01
影响因子：
5.5
通讯作者：
Stutts, MJ
中科院分区：
文献类型：
--
作者：
Caldwell, RA;Boucher, RC;Stutts, MJ
上皮Na+通道（ENaC）功能的调节对正常的盐和水平衡至关重要。这种调节是通过细胞表面通道的插入/恢复、通道打开概率（P-o）的变化或这些过程的组合来实现的。上皮源丝氨酸蛋白酶，包括通道激活蛋白酶（CAP）和前列腺蛋白酶，调节上皮Na+运输，但分子机制尚不清楚。我们测试了细胞外丝氨酸蛋白酶激活居住在质膜上的近乎沉默的ENaC种群的假设。在暴露于胰蛋白酶（一种与CAP和前列腺蛋白酶同源的丝氨酸蛋白酶）之前和期间，在稳定表达大鼠α， β和γ亚基（rENaC）的成纤维细胞（NIH/3T3）的外-外斑块中记录单通道事件。在基线条件下，近沉默斑块被定义为rENaC活性(NPo) < 0.03，其中N为通道数。3 μ g/ml胰蛋白酶灌注1 ~ 5min， NPo增加66倍（n = 7）。在观察到含有单一功能通道的斑块中，胰蛋白酶将P-o从0.02 +/- 0.01增加到0.57 +/- 0.03 (n = 3，平均+/- SE)，这是由于通道打开时间增加和通道关闭时间减少的共同作用。近沉默ENaC的活化需要催化活性。通道电导和Na+/ Li+电流比与胰蛋白酶相似。内源性上皮丝氨酸蛋白酶对ENaC P-o的调节是上皮Na+运输的潜在重要调节剂，不同于激素诱导的质膜插入通道的调节。
The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/ retrieval of channels, by changes in channel open probability (P-o), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat alpha-, beta-, and gamma-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity (NPo) < 0.03, where N is the number of channels. Within 1 - 5 min of 3 mu g/ml bath trypsin superfusion, NPo increased similar to 66-fold ( n = 7). In patches observed to contain a single functional channel, trypsin increased P-o from 0.02 +/- 0.01 to 0.57 +/- 0.03 ( n = 3, mean +/- SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/ Li+ current ratio with trypsin were similar to control values. Modulation of ENaC P-o by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels.