Quantitative analysis of mammalian GIRK2 channel regulation by G proteins, the signaling lipid PIP2 and Na+ in a reconstituted system.
Quantitative analysis of mammalian GIRK2 channel regulation by G proteins, the signaling lipid PIP2 and Na+ in a reconstituted system.
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DOI：
10.7554/elife.03671
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发表时间：
2014-07-20
期刊：
影响因子：
7.7
通讯作者：
MacKinnon R
中科院分区：
文献类型：
--
作者：
Wang W;Whorton MR;MacKinnon R
GIRK 通道控制心房起搏细胞的尖峰频率和神经元的抑制电位。通过直接响应 G 蛋白、PIP2 和 Na+，GIRK 受到多种信号通路的控制。在这项研究中，哺乳动物 GIRK2 通道已在平面脂质膜中纯化和重建，并分析了 Gα、Gβγ、PIP2 和 Na+ 的影响。 Gβγ 和 PIP2 必须同时存在才能激活 GIRK2。 Na+ 不是必需的，但可以调节 Gβγ 和 PIP2 在生理浓度上的作用。 Gαi1(GTPγS) 没有影响，而 Gαi1(GDP) 通过去除 Gβγ 来关闭通道。在 Gβγ 存在的情况下，GIRK2 作为 PIP2 摩尔分数的函数打开，希尔系数为 2.5，并且具有使 GIRK2 能够响应 PIP2 浓度自然变化的亲和力。 Gβγ 和 PIP2 的双重要求有助于解释为什么 GIRK2 被 Gi/o 激活，而不是 Gq 偶联的 GPCR。 DOI：http://dx.doi.org/10.7554/eLife.03671.001 虽然体内的每个细胞都被膜包围，但分子可以通过多种方式穿过该膜进入或离开细胞。 GIRK 家族的蛋白质在哺乳动物细胞膜中形成通道，当这些通道打开时，钾离子可以流过膜以控制膜的电压。 GIRK 通道存在于心脏和中枢神经系统中，可以通过多种方式激活。钠离子和称为“信号脂质”的分子可以调节 GIRK 通道的激活。这些通道也可以由 G 蛋白打开：G 蛋白存在于细胞内部，有助于将信号从细胞外部传输到内部。三种 G 蛋白（称为 Gα、Gβ 和 Gγ）在一个复合体中协同工作，其功能有点像开关。当打开时，Gα 亚基与其他两个亚基（称为 Gβγ）分离；然后这两个部分都可以激活细胞内不同的信号通路。多年来，人们已知 Gβγ 亚基和信号脂质可以调节 GIRK 通道的开放，但这些事件仅在活细胞的背景下进行了研究。每个分子的具体作用以及 Gα 亚基是否也可以调节 GIRK 通道仍然未知。现在王等人。在酵母细胞中产生了一种名为 GIRK2 的小鼠 GIRK 通道，纯化了这种蛋白质，并将其添加到人造膜中。这种“重构系统”允许在比以前的实验更受控的条件下研究 GIRK 通道的调节。王等人。发现 Gβγ 亚基和信号脂质都需要存在才能激活 GIRK2 通道。钠离子不是必需的，但当 Gβγ 和信号脂质已经存在时，钠离子会促进进一步开放。当锁定在“开启”状态时，Gα 亚基对 GIRK2 没有影响，但添加锁定在“关闭”状态的 Gα 可以通过去除 Gβγ 蛋白来关闭这些通道。王等人的研究结果。建议应该可以使用类似的重构系统来研究是什么允许不同的 G 蛋白激活特定的信号通路。 DOI：http://dx.doi.org/10.7554/eLife.03671.002
GIRK channels control spike frequency in atrial pacemaker cells and inhibitory potentials in neurons. By directly responding to G proteins, PIP2 and Na+, GIRK is under the control of multiple signaling pathways. In this study, the mammalian GIRK2 channel has been purified and reconstituted in planar lipid membranes and effects of Gα, Gβγ, PIP2 and Na+ analyzed. Gβγ and PIP2 must be present simultaneously to activate GIRK2. Na+ is not essential but modulates the effect of Gβγ and PIP2 over physiological concentrations. Gαi1(GTPγS) has no effect, whereas Gαi1(GDP) closes the channel through removal of Gβγ. In the presence of Gβγ, GIRK2 opens as a function of PIP2 mole fraction with Hill coefficient 2.5 and an affinity that poises GIRK2 to respond to natural variations of PIP2 concentration. The dual requirement for Gβγ and PIP2 can help to explain why GIRK2 is activated by Gi/o, but not Gq coupled GPCRs. DOI: http://dx.doi.org/10.7554/eLife.03671.001 Though every cell in the body is surrounded by a membrane, there are a number of ways that molecules can pass through this membrane to either enter or leave the cell. Proteins from the GIRK family form channels in the membranes of mammalian cells, and when open these channels allow potassium ions to flow through the membrane to control the membrane's voltage. GIRK channels are found in the heart and in the central nervous system, and can be activated in a variety of ways. Sodium ions and molecules called ‘signaling lipids’ can regulate the activation of GIRK channels. These channels can also be caused to open by G proteins: proteins that are found inside cells and that help to transmit signals from the outside of a cell to the inside. Three G proteins—called Gα, Gβ, and Gγ—work together in a complex that functions a bit like a switch. When switched on, the Gα subunit is separated from the other two subunits (called Gβγ); and both parts can then activate different signaling pathways inside the cell. The Gβγ subunits and a signaling lipid have been known to regulate the opening of GIRK channels for a number of years, but these events have only been studied in the context of living cells. The specific role of each molecule, and whether the Gα subunit can also regulate the GIRK channels, remains unknown. Now Wang et al. have produced one type of mouse GIRK channel, called GIRK2, in yeast cells, purified this protein, and added it into an artificial membrane. This ‘reconstituted system’ allowed the regulation of a GIRK channel to be investigated under more controlled conditions than in previous experiments. Wang et al. found that the Gβγ subunits and the signaling lipid both need to be present to activate the GIRK2 channel. Sodium ions were not essential, but promoted further opening when Gβγ and the signaling lipid were already present. When locked in its ‘on’ state, the Gα subunit had no effect on GIRK2, but adding Gα locked in the ‘off’ state closed these channels by removing the Gβγ proteins. The findings of Wang et al. suggest that it should be possible to use a similar reconstituted system to investigate what allows different G proteins to activate specific signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.03671.002