Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool.
Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool.
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从回收通道库中的胞外插入介导肾细胞系中cAMP刺激的急性ENAC刺激。
DOI：
10.1085/jgp.200409124
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发表时间：
2005-01
期刊：
影响因子：
--
通讯作者：
Frizzell RA
中科院分区：
文献类型：
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作者：
Butterworth MB;Edinger RS;Johnson JP;Frizzell RA
致密上皮细胞上皮钠通道(ENaC)的急性激素调节通过细胞内通道向根尖表面的运输增加了跨细胞的Na+转运。激动剂冲刷后从根尖表面移除的通道的命运还不是很清楚。通过重复刺激极化的小鼠皮质集合管(MCCD，MPKCCD14)上皮细胞，我们评估了ENaC通过细胞内池循环可重新插入根尖膜的假设。用改良Ussing小室记录MCCD上皮细胞的短路电流(ISC)、膜电容(CT)和电导(GT)。ENaC的表面生物素化显示，cAMP刺激后，顶膜上的通道数量增加。其中ISC增加83±6%(n=31)，CT增加15.3±1.5%(n=15)。选择性膜通透性试验表明，CT升高是由于顶膜电容增加所致。ISC和CT在刺激消失时降至基础水平。重复的cAMP刺激和清洗(∼，每个周期1h)导致反应疲劳，每个刺激恢复周期，ΔISC使∼下降10%。当通道产生被放线菌酮阻断时，ΔISC在每个刺激周期中减少∼15%，这表明新合成的ENaC贡献了动员到顶膜的通道的相对较小的部分。用苯扎米选择性阻断表面ENaC表明，从根尖下池插入的通道占刺激后ISC的90%，在重新刺激时，从根尖表面取回的通道有很大比例重新插入根尖膜。通道循环被抑制ENaC胞吐的布雷菲尔丁A、抑制ENaC内吞和循环的氯喹以及阻断ENaC胞吐的Latrunculin A破坏。以顶膜和细胞内循环池中的通道种群为特征的隔室模型为ISC对重复刺激的反应提供了足够的动力学描述。该模型支持ENaC循环以响应重复的cAMP刺激的概念。
Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.