Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.
Fatty acid modulation and polyamine block of GluK2 kainate receptors analyzed by scanning mutagenesis.
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通过扫描诱变分析 GluK2 红藻氨酸受体的脂肪酸调节和多胺阻断。
DOI：
10.1085/jgp.201010442
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Huettner,JamesE
中科院分区：
文献类型：
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作者：
Wilding,TimothyJ;Chen,Kevin;Huettner,JamesE
Q/R位点红藻氨酸受体亚单位的RNA编辑决定了它们对顺式不饱和脂肪酸的抑制以及细胞质多胺的阻断的敏感性。由未编辑(Q)亚基组成的通道被多胺强烈阻断，但对脂肪酸不敏感，如花生四烯酸(AA)和二十二碳六烯酸(DHA)，而同源编辑(R)通道抵抗多胺阻断，但被AA和DHA抑制。在本研究中，我们分析了同源重组GluK2(以前的GluR6)通道介导的脂肪酸对全细胞电流的调节，孔环中的单个残基，扫描突变取代了M1和M3跨膜螺旋。我们的结果定义了沿着M1、M2和M3螺旋的三个毗邻表面，在这些表面上，功能增益取代使GluK2(Q)通道容易受到脂肪酸抑制。此外，我们在M3螺旋上的中央空洞水平确定了四个位置(F611、L614、S618和T621)，在这些位置上，Arg替代增加了对氯的相对通透性，消除了多胺阻断。值得注意的是，对于其中两个位置，L614R和S618R，暴露在脂肪酸中会降低表观氯离子渗透性，并分别增强全细胞电流∼5倍和2.5倍。综上所述，我们的结果表明，AA和DHA改变了处于开放状态的M3的取向，这取决于M1、M2和M3之间界面的接触。此外，我们的结果表明，尽管与钾通道和其他孔环家族成员相比，GluK2的取向相反，但中央空腔内的侧链在决定离子选择性和细胞质多胺的阻断方面具有重要意义。
RNA editing of kainate receptor subunits at the Q/R site determines their susceptibility to inhibition by cis-unsaturated fatty acids as well as block by cytoplasmic polyamines. Channels comprised of unedited (Q) subunits are strongly blocked by polyamines, but insensitive to fatty acids, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), whereas homomeric edited (R) channels resist polyamine block but are inhibited by AA and DHA. In the present study, we have analyzed fatty acid modulation of whole-cell currents mediated by homomeric recombinant GluK2 (formerly GluR6) channels with individual residues in the pore-loop, M1 and M3 transmembrane helices replaced by scanning mutagenesis. Our results define three abutting surfaces along the M1, M2, and M3 helices where gain-of-function substitutions render GluK2(Q) channels susceptible to fatty acid inhibition. In addition, we identify four locations in the M3 helix (F611, L614, S618, and T621) at the level of the central cavity where Arg substitution increases relative permeability to chloride and eliminates polyamine block. Remarkably, for two of these positions, L614R and S618R, exposure to fatty acids reduces the apparent chloride permeability and potentiates whole-cell currents ∼5 and 2.5-fold, respectively. Together, our results suggest that AA and DHA alter the orientation of M3 in the open state, depending on contacts at the interface between M1, M2, and M3. Moreover, our results demonstrate the importance of side chains within the central cavity in determining ionic selectivity and block by cytoplasmic polyamines despite the inverted orientation of GluK2 as compared with potassium channels and other pore-loop family members.