Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane
Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane
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DOI：
10.1074/jbc.m409462200
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发表时间：
2005-02-04
影响因子：
4.8
通讯作者：
Foskett, JK
中科院分区：
文献类型：
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作者：
Ferreri-Jacobia, M;Mak, DOD;Foskett, JK
肌醇1,4,5-三磷酸受体（InsP(3)R）是内质网中的一种完整的膜蛋白。（ER）作为配体门控的Ca2+释放通道。InsP(3)介导的Ca2+释放调节胞质游离Ca2+浓度（[Ca2+](i)），提供普遍存在的细胞内信号，具有高时间和空间特异性。InsP，R的精确定位被认为对于提供局部[Ca2+]调节和确保Ca2+释放位点之间的有效功能耦合是重要的，在面对Ca2+诱导的Ca2+释放的本质不稳定的再生过程时，通过增加刺激强度来实现通道的分级招募。高度局部化的Ca2+释放归因于InsP3R通道聚集和定位于离散区域的能力，这表明可能存在限制其运动的机制。在这里，我们检测了InsP(3)R （InsP(3)R3）的3型异构体（InsP(3)R3）在ER膜中的横向迁移率，方法是在InsP(3)R3与绿色荧光蛋白融合到其N端光漂白后进行共聚焦荧光恢复。在中国仓鼠卵巢和COS-7细胞中，室温下的扩散系数D接近4 × 10(-10) cm(2)/s，这一数值与其他er定位的整体膜蛋白相似，通道的移动率很高（接近75%）。D在37°c时适度增加，并且由于ATP的消耗，它以及可移动分数可逆地降低。虽然破坏肌动蛋白细胞骨架（latrunculin）没有影响，但破坏微管（nocodazole）使D减少一半而不影响可移动部分。我们得出结论，整个内质网在这些细胞中是连续的，绝大多数InSP(3)R3通道可以自由地扩散穿过它，其速率与其他多面体内质网整体膜蛋白的速率相当。观察到的InsP(3)R3迁移率可能高于其固有的扩散迁移率，因为额外的atp和微管促进了通道的迁移。
The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an integral membrane protein in the endoplasmic reticulum. (ER) which functions as a ligand-gated Ca2+ release channel. InsP(3)-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+](i)), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP,R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP(3)R (InsP(3)R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InSP(3)R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was similar to4 x 10(-10) cm(2)/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (similar to75%) of channels mobile. D was modestly increased at 37 degreesC, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption,of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InSP(3)R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP(3)R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATPand microtubule-facilitated motility of the channel.