Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids.
Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids.
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DOI：
10.1371/journal.pone.0023886
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Zeilhofer HU
中科院分区：
文献类型：
--
作者：
Yévenes GE;Zeilhofer HU
甘氨酸受体（GlyR）是Cys环超家族的递质门控阴离子通道，其介导脊髓和脊髓上位点的突触抑制。尽管它们在运动控制和感觉处理中发挥关键作用，但它们尚未被开发为药物靶点，部分原因是迄今为止变构控制的可能性有限。内源性大麻素（ECs）最近已被定性为直接变构GlyR调节剂，但潜在的分子位点仍然未知。在这里，我们表明，化学中性EC（如花生四烯酸，AEA）是α1，α2和α3 GlyRs的正调节剂，而酸性EC（如N-花生四烯酸-甘氨酸; NA-Gly）增强α1 GlyRs，但抑制α2和α3。这种亚基特异性使我们能够通过分析嵌合和突变受体来确定潜在的分子位点。我们发现细胞外环2的丙氨酸52、跨膜（TM）区2的甘氨酸254和细胞内赖氨酸385决定NA-Gly对α1 GlyRs的正性调节。α2中非保守的细胞外和TM残基的连续取代将NA-Gly介导的抑制转化为增强。相反，在TM 3和TM 4之间的细胞内环内保守赖氨酸的突变减弱了NA-Gly介导的α1 GlyRs增强作用，而不影响α2和α3的抑制作用。值得注意的是，这种突变减少了所有三种GlyR的AEA调节。这些结果定义了EC对GlyRs进行变构控制的分子位点，并揭示了TM 3 -4胞内环在Cys环离子通道变构调节中的未识别功能。这些网站的识别可能有助于了解这种调制的生理作用，并促进疾病，如痉挛，惊吓疾病和可能的慢性疼痛的新的治疗方法的发展。
Glycine receptors (GlyRs) are transmitter-gated anion channels of the Cys-loop superfamily which mediate synaptic inhibition at spinal and selected supraspinal sites. Although they serve pivotal functions in motor control and sensory processing, they have yet to be exploited as drug targets partly because of hitherto limited possibilities for allosteric control. Endocannabinoids (ECs) have recently been characterized as direct allosteric GlyR modulators, but the underlying molecular sites have remained unknown. Here, we show that chemically neutral ECs (e.g. anandamide, AEA) are positive modulators of α1, α2 and α3 GlyRs, whereas acidic ECs (e.g. N-arachidonoyl-glycine; NA-Gly) potentiate α1 GlyRs but inhibit α2 and α3. This subunit-specificity allowed us to identify the underlying molecular sites through analysis of chimeric and mutant receptors. We found that alanine 52 in extracellular loop 2, glycine 254 in transmembrane (TM) region 2 and intracellular lysine 385 determine the positive modulation of α1 GlyRs by NA-Gly. Successive substitution of non-conserved extracellular and TM residues in α2 converted NA-Gly-mediated inhibition into potentiation. Conversely, mutation of the conserved lysine within the intracellular loop between TM3 and TM4 attenuated NA-Gly-mediated potentiation of α1 GlyRs, without affecting inhibition of α2 and α3. Notably, this mutation reduced modulation by AEA of all three GlyRs. These results define molecular sites for allosteric control of GlyRs by ECs and reveal an unrecognized function for the TM3-4 intracellular loop in the allosteric modulation of Cys-loop ion channels. The identification of these sites may help to understand the physiological role of this modulation and facilitate the development of novel therapeutic approaches to diseases such as spasticity, startle disease and possibly chronic pain.
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