McNicholas C M, Wang W, Ho K, Hebert S C, Giebisch G
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510.
Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8077-81. doi: 10.1073/pnas.91.17.8077.
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 Mg(2+)-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 microM) and calyculin A (1 microM) 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 Mg(2+)-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 Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-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.
最近从大鼠肾脏中克隆出一种具有独特分子结构的内向整流型、ATP调节的钾通道——ROMK1。我们使用膜片钳技术研究了ROMK1的调节，特别关注磷酸化/去磷酸化过程。在存在Mg2+的情况下，将膜片切除到无ATP的浴液中后，通道会自发衰减。将膜片切除到无Mg(2+)的浴液中，或在存在Mg2+的情况下用广谱磷酸酶抑制剂原钒酸盐预孵育后，通道衰减几乎完全消除。MgATP预孵育也以剂量依赖的方式抑制通道衰减。此外，还研究了特异性磷酸酶抑制剂冈田酸（1 microM）和花萼海绵诱癌素A（1 microM）的作用。冈田酸或花萼海绵诱癌素A的存在均未能抑制通道衰减。综上所述，这些数据表明ROMK1的衰减涉及Mg(2+)依赖的去磷酸化过程。向浴液中添加MgATP后，通道活性也部分恢复。添加外源性cAMP依赖性蛋白激酶A（PKA）催化亚基导致通道开放概率进一步增加。在不存在Mg2+的情况下添加Na2ATP无效，这表明通道活性的恢复是一个Mg(2+)依赖的过程。向浴液中添加特异性PKA抑制剂PKI会导致通道活性部分、可逆的抑制。因此，PKA依赖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磷酸化位点一致。
