Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate
Regulation of Kv7 (KCNQ) K+ channel open probability by phosphatidylinositol 4,5-bisphosphate
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DOI：
10.1523/jneurosci.2597-05.2005
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发表时间：
2005-10-26
影响因子：
5.3
通讯作者：
Shapiro, MS
中科院分区：
文献类型：
--
作者：
Li, Y;Gamper, N;Shapiro, MS
电压门控Kv 7（KCNQ）通道是重要的K+电流（包括神经元M电流）的基础，并且被认为对膜磷脂酰肌醇4，5-二磷酸（PIP 2）和PIP 2消耗敏感，从而成为毒蕈碱受体抑制的基础。我们在中国仓鼠卵巢（CHO）细胞上，使用单通道和全细胞膜片钳和生化分析研究了PIP 2对Kv7.2-7.4通道的调节。发现Kv7.2-Kv7.4同源多聚体和Kv7.2/7.3异源多聚体的最大开放概率（P-o）强烈依赖于应用于由内而外补丁的[diC 8-PIP 2]，具有与它们在on-cell模式中的最大P-o相关的差异表观亲和力。单位电导不受PIP 2的影响。通过磷脂酰肌醇（4）5-激酶的共表达提高强直性[PIP 2]增加了在细胞贴片中研究的Kv7.2和Kv7.2/7.3通道的最大P-o，并增加了全细胞Kv7.2，但不是Kv7.3，电流幅度。在与毒蕈碱M-1受体共表达的细胞中，毒蕈碱激动剂的浴应用降低了在细胞上贴片中分离的Kv7.2/7.3通道的最大P-o。共表达的PIP 2螯合构建体适度降低全细胞Kv7.2/7.3电流，共表达的构建体含有PIP 2磷酸酶几乎废除了它们。最后，在CHO细胞中稳定表达M-1受体的阴离子磷脂的生化分析表明，PIP 2和PIP几乎耗尽毒蕈碱刺激后1分钟，与一个意想不到的反弹后10分钟。这些结果强烈支持的直接调节Kv 7通道的PIP 2和它的消耗作为M通道的毒蕈碱抑制的机制。Kv7.2-7.4通道对PIP 2的不同表观亲和力可能是在细胞附着的斑块中观察到的高度差异的最大P-o的基础。
Voltage-gated Kv7 (KCNQ) channels underlie important K+ currents, including the neuronal M current, and are thought to be sensitive to membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2 depletion to underlie muscarinic receptor inhibition. We studied regulation of Kv7.2-7.4 channels by PIP2 in Chinese hamster ovary (CHO) cells using single-channel and whole-cell patch clamp and biochemical analysis. Maximal open probabilities (P-o) of Kv7.2-Kv7.4 homomultimers and of Kv7.2/7.3 heteromultimers were found to be strongly dependent on the [diC8-PIP2] applied to inside-out patches, with differential apparent affinities that correlate with their maximal P-o in on-cell mode. Unitary conductance was not affected by PIP2. Raising tonic [PIP2] by coexpression of phosphatidylinositol (4) 5-kinase increased the maximal P-o of both Kv7.2 and Kv7.2/7.3 channels studied in on-cell patches and increased whole-cell Kv7.2, but not Kv7.3, current amplitudes. In cells coexpressed with muscarinic M-1 receptors, bath application of muscarinic agonist reduced the maximal P-o of Kv7.2/7.3 channels isolated in on-cell patches. Coexpression of a PIP2 sequestering construct moderately reduced whole-cell Kv7.2/7.3 currents, and coexpression of a construct containing a PIP2 phosphatase nearly abolished them. Finally, biochemical analysis of anionic phospholipids in CHO cells stably expressing M-1 receptors shows that PIP2 and PIP are nearly depleted 1 min after muscarinic stimulation, with an unexpected rebound after 10 min. These results strongly support the direct regulation of Kv7 channels by PIP2 and its depletion as the mechanism of muscarinic suppression of M channels. Divergent apparent affinities of Kv7.2-7.4 channels for PIP2 may underlie their highly differential maximal P-o observed in cell-attached patches.