Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors
Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors
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DOI：
10.1074/jbc.m112.415786
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发表时间：
2012-12-21
影响因子：
4.8
通讯作者：
Joseph, Suresh K.
中科院分区：
文献类型：
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作者：
Bhanumathy, Cunnigaiper;da Fonseca, Paula C. A.;Joseph, Suresh K.
我们已经结合丙氨酸诱变和功能测定，以确定在通道结构域中的氨基酸残基是至关重要的肌醇1，4，5-三磷酸受体（IP 3R）通道功能。选择的残基在所有三种IP 3R亚型中高度保守，并且位于S6孔衬螺旋的胞质末端和C-尾的近端部分。两个相邻的疏水性氨基酸（Ile-2588和Ile-2589）在假定的胞质界面的S6螺旋失活通道功能，并可能是候选人的通道门。五个带负电荷的残基突变，没有完全消除通道功能。在突变的五个带正电荷的残基中，只有一个使通道失活（Arg-2596）。除了先前确定的C-尾中一对半胱氨酸（Cys-2610和Cys-2613）的作用外，一对高度保守的组氨酸（His-2630和His-2635）也是通道功能所必需的。H2630 A和H2635 A突变体（但不是R2596 A）的表达产生了N末端片段和通道结构域之间相互作用不稳定的受体。使用GST下拉测定证明了胞质C-尾和TM 4，5-环之间的先前未识别的关联。然而，C-尾中的突变均不干扰这种相互作用或改变C-尾组装成二聚体的能力。我们目前的研究结果和最近的信息IP 3R结构的电子显微镜和晶体学被纳入一个修订后的模型通道门控。
We have combined alanine mutagenesis and functional assays to identify amino acid residues in the channel domain that are critical for inositol 1,4,5-trisphosphate receptor (IP3R) channel function. The residues selected were highly conserved in all three IP3R isoforms and were located in the cytosolic end of the S6 pore-lining helix and proximal portion of the C-tail. Two adjacent hydrophobic amino acids (Ile-2588 and Ile-2589) at the putative cytosolic interface of the S6 helix inactivated channel function and could be candidates for the channel gate. Of five negatively charged residues mutated, none completely eliminated channel function. Of five positively charged residues mutated, only one inactivated the channel (Arg-2596). In addition to the previously identified role of a pair of cysteines in the C-tail (Cys-2610 and Cys-2613), a pair of highly conserved histidines (His-2630 and His-2635) were also essential for channel function. Expression of the H2630A and H2635A mutants (but not R2596A) produced receptors with destabilized interactions between the N-terminal fragment and the channel domain. A previously unrecognized association between the cytosolic C-tail and the TM 4,5-loop was demonstrated using GST pull-down assays. However, none of the mutations in the C-tail interfered with this interaction or altered the ability of the C-tail to assemble into dimers. Our present findings and recent information on IP3R structure from electron microscopy and crystallography are incorporated into a revised model of channel gating.