Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels.
Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels.
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DOI：
10.1085/jgp.201511354
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发表时间：
2015-05
期刊：
影响因子：
--
通讯作者：
Gordon SE
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文献类型：
--
作者：
Ufret-Vincenty CA;Klein RM;Collins MD;Rosasco MG;Martinez GQ;Gordon SE
磷酸肌醇结合到近端C-末端区域的选择性位点以调节TRPV 1。尽管PI（4，5）P2被认为在调节许多离子通道和转运蛋白的活性中起重要作用，但其机制尚不清楚。在这里，我们利用TRPV 1离子通道区分PI（4，5）P2和PI（4）P的能力来定位TRPV 1序列中与磷酸肌醇直接相互作用的区域。我们在TRP盒R721 A后的近端C-末端区域发现了一个点突变，该点突变逆转了TRPV 1的选择性。尽管R721 A突变仅使PI（4，5）P2激活的EC 50增加30%，但它使PI（4）P激活的EC 50降低了两个数量级以上。我们使用化学诱导的和电压激活的磷酸酶来确定PI（4）P继续支持TRPV 1活性，即使在PI（4，5）P2从质膜耗尽之后。我们的数据不能解释一个纯粹的静电机制之间的相互作用的磷酸肌醇和蛋白质，类似的MARCKS（豆蔻酰丙氨酸丰富的C激酶底物）效应域或EGF受体。相反，PI（4，5）P2-选择性通道转化为PI（4）P-选择性通道表明结构化的磷酸肌醇结合位点介导TRPV 1活性的调节，并且721位的氨基酸可能与磷酸肌醇5′位的部分直接相互作用。
Phosphoinositides bind to a selective site in the proximal C-terminal region to regulate TRPV1. Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5′ position of the phosphoinositide.
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影响因子：
16.2
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