REGULATION OF ROMK1 K+ CHANNEL ACTIVITY INVOLVES PHOSPHORYLATION PROCESSES
REGULATION OF ROMK1 K+ CHANNEL ACTIVITY INVOLVES PHOSPHORYLATION PROCESSES
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DOI：
10.1073/pnas.91.17.8077
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发表时间：
1994-08-16
影响因子：
11.1
通讯作者：
GIEBISCH, G
中科院分区：
文献类型：
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作者：
MCNICHOLAS, CM;WANG, WH;GIEBISCH, G
最近从大鼠肾脏中克隆了一个具有独特分子结构的内向整流、受ATP调控的K+通道ROMK1。用膜片钳技术，我们研究了ROMK1的调节，特别是磷酸化/去磷酸化过程，在有镁离子存在的无ATP的浴液中，膜片切除后发生了自发的通道衰退。在没有镁离子的浴液中切除斑块或在有镁离子存在的情况下与广谱磷酸酶抑制剂原钒酸预孵育后，几乎完全消除了通道衰竭。镁ATP预孵育也以剂量依赖的方式抑制通道耗竭。此外，还研究了特异性磷酸酶抑制剂冈田酸(1 MU M)和花盏花素A(1 MU M)的作用，发现冈田花酸或花盏花素A均不能抑制通道衰弱。综上所述，这些数据表明ROMK1的降解涉及到一个依赖于镁离子的去磷酸化过程。加入镁三磷酸腺苷后，通道活动也部分恢复。外源性cAMP依赖的蛋白激酶A(PKA)催化亚基的加入进一步增加了通道的开放概率。Na(2)-ATP的加入在没有镁离子的情况下无效，这表明通道活动的恢复是一个镁离子依赖的过程。在镀液中加入特定的PKA抑制剂PKI，可部分、可逆地抑制通道活性。因此，依赖于PKA的磷酸化过程参与了通道活性的调节。这一观察结果与ROMK1上存在潜在的PKA磷酸化位点是一致的。
An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes, Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg2+-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 mu M) and calyculin A (1 mu M) was also investigated, The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg2+-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na(2)ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg2+-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.