Department of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, London WC1E 6BT, United Kingdom.
Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
J Biol Chem. 2021 Jan-Jun;296:100387. doi: 10.1074/jbc.RA119.012358. Epub 2021 Feb 20.
Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyR, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyR are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyR (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyR converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.
与其他五聚体配体门控通道一样，甘氨酸受体（GlyRs）在跨膜螺旋 3 和 4 之间包含长的细胞内结构域（ICD）。结构上表征的 GlyRs 通常被设计为具有非常短的 ICD。我们在这里展示，对于这样的一种构建体，斑马鱼 GlyR，激动剂甘氨酸、β-丙氨酸、牛磺酸和 GABA 具有高功效，并产生大于 0.9 的最大单通道开放概率。相比之下，对于全长人α1 GlyR，牛磺酸和 GABA 显然是部分激动剂，最大开放概率分别为 0.46 和 0.09。我们发现，GlyR 中升高的开放概率不是由于人源和斑马鱼同源物之间的有限序列差异，而是由于用短三肽 ICD 替换了天然 ICD。与这一解释一致的是，缩短人 GlyR 的 ICD 会增加牛磺酸和 GABA 产生的最大开放概率，分别增加到 0.90 和 0.70，但进一步将其工程化使其类似于 GlyR（通过引入斑马鱼跨膜螺旋 4 和 C 末端）没有效果。此外，将天然 ICD 重新引入 GlyR 将牛磺酸和 GABA 转化为部分激动剂，最大开放概率分别为 0.66 和 0.40。短和长 ICD GlyR 亚基的跨膜螺旋 3 和 4 的结构比较表明，ICD 缩短不会扭曲每个亚基内这些螺旋的取向。这表明 ICD 缩短的影响源于去除天然 ICD 对 GlyR 门控的调节作用，揭示了 ICD 在五聚体配体门控通道中的新作用。
