Activation of Na+-permeant cation channel by stretch and cyclic AMP-dependent phosphorylation in renal epithelial A6 cells.
Activation of Na+-permeant cation channel by stretch and cyclic AMP-dependent phosphorylation in renal epithelial A6 cells.
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DOI：
10.1085/jgp.110.3.327
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发表时间：
1997-09
期刊：
影响因子：
--
通讯作者：
Niisato N
中科院分区：
文献类型：
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作者：
Marunaka Y;Shintani Y;Downey GP;Niisato N
目前认为，非选择性阳离子 (NSC) 通道对精氨酸催产素（一种抗利尿激素）和拉伸作出反应，调节远端肾单位的 Na+ 吸收。然而，该通道的调节机制仍未完全表征。为了研究该通道的调节机制，我们使用了在渗透性支持物上培养的肾上皮细胞（A6）。 A6 细胞汇合单层的顶膜表达 29-pS 通道，该通道通过拉伸或磷酸二酯酶抑制剂 3-异丁基-1-甲基黄嘌呤 (IBMX) 激活。该通道对 Na+、K+、Li+ 和 Cs+ 具有相同的选择性，但对 Ca2+ (PCa/PNa < 0.005) 或 Cl− (PCl/PNa < 0.01) 几乎没有选择性，将其识别为 NSC 通道。拉伸对 IBMX 激活通道的开放概率 (P o) 没有额外影响。该通道在基础、拉伸或 IBMX 刺激条件下具有一种开放 (“O”) 状态和两种闭合状态（短“C S”和长“C L”）状态。拉伸和IBMX都增加了通道的P o ，而平均打开或关闭时间没有任何可检测到的变化。这些观察结果使我们得出这样的结论：动力学模型“C L ↔ C S ↔ O”是三种可能的线性模型中最合适的。根据该模型，IBMX 或拉伸会降低 C L 从 C S 的通道离开率，导致 P o 增加。细胞松弛素 D 预处理消除了对拉伸或 IBMX 的反应，但不改变基础活性。 H89（cAMP 依赖性蛋白激酶抑制剂）完全消除了对拉伸和 IBMX 的反应，但与细胞松弛素 D 不同，它还降低了基础活性。我们的结论是：(a) cAMP 激活的 NSC 通道的功能特性与拉伸激活的 NSC 通道相似，(b) 肌动蛋白细胞骨架在拉伸和 cAMP 诱导的 NSC 通道激活中起着至关重要的作用，(c) NSC 通道的基础活性由 PKA 依赖性磷酸化维持，但不依赖于肌动蛋白微丝。
It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na+ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na+, K+, Li+, and Cs+, but little selectivity for Ca2+ (PCa/PNa < 0.005) or Cl− (PCl/PNa < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (P o) of the IBMX-activated channel. This channel had one open (“O”) and two closed (short “C S” and long “C L”) states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the P o of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model “C L ↔ C S ↔ O” was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for C L from C S, resulting in an increase in P o. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.