Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag*
Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag*
复制标题
使用 α-银环蛇毒素标签追踪细胞表面 GABAB 受体*
DOI：
10.1074/jbc.m803197200
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发表时间：
2008
影响因子：
4.8
通讯作者：
T. Smart
中科院分区：
文献类型：
--
作者：
M. Wilkins;Xinyan Li;T. Smart
GABAB 受体介导中枢神经系统中的缓慢突触抑制，对于突触可塑性非常重要，并且与疾病有关。 GABAB 受体位于突触前和突触后位点，会影响细胞兴奋性，但其有效性部分取决于它们向细胞表面膜的运输及其稳定性。为了检查 GIRK 细胞和神经元中 GABAB 受体的动态行为，我们设计了一种方法，该方法基于用神经毒素 α-银环蛇毒素的结合位点成分标记受体。通过使用与 R2 亚基共表达的 α-银环蛇毒素结合位点标记的 GABAB R1a 亚基 (R1aBBS)，我们可以使用小报告分子、α-银环蛇毒素缀合的罗丹明来追踪受体的迁移性。通过这种方式，可以用固定细胞和活细胞测量这些受体的内化和膜插入速率。结果表明，GABAB受体在细胞膜中快速周转，内化速率受受体激活状态的影响。基于金环蛇毒素的受体标记方法似乎非常适合跟踪其他 G 蛋白偶联受体的动态调节。
GABAB receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABAB receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABAB receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, α-bungarotoxin. By using the α-bungarotoxin binding site-tagged GABAB R1a subunit (R1aBBS), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, α-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABAB receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors.