School of Biomedical Sciences and Pharmacy, University of Newcastle and Hunter Medical Research Institute, Newcastle, NSW 2308, Australia.
J Physiol. 2011 May 15;589(Pt 10):2459-74. doi: 10.1113/jphysiol.2011.206326. Epub 2011 Mar 8.
Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of α and β subunits assembled in a 2:3 stoichiometry. The α1/βheteromer is considered the dominant GlyR isoform at 'native' adult synapses in the spinal cord and brainstem. However, the α3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I-II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the α1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH.We usedwhole cell recording (at 22-24◦C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg⁻¹, I.P.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic micewere reduced by 25% and 50%, respectively (42.0 ± 3.6 pA vs. 31.0 ± 1.8 pA, P <0.05 and 7.4 ± 0.5 ms vs. 5.0 ± 0.4 ms, P <0.05; means ± SEM, n =34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABAAergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50 =130 ± 20 μM vs. 64 ± 11 μM, respectively; n =8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of α1-containing GlyRs in SDHneurones.Glycine sensitivity, based on the response to 1-3 μM glycine, was reduced in>75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design therapies that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.
抑制性甘氨酸受体 (GlyRs) 是由 α 和 β 亚基组成的五聚体配体门控离子通道，以 2:3 的化学计量比组装。α1/β 异质体被认为是脊髓和脑干中“天然”成年突触上的主要 GlyR 同工型。然而，α3 GlyR 亚基集中在浅层背角 (SDH：I-II 层)，这是一个对来自皮肤、肌肉和内脏的伤害性信号进行处理的脊髓区域。在这里，我们使用痉挛性小鼠，其 GlyR 的 α1 亚基中存在自然发生的突变 (A52S)，来研究突变对抑制性突触传递和同型稳态可塑性的影响，并探测 SDH 中各种 GlyR 亚基的存在。我们使用全细胞膜片钳记录（在 22-24°C 下），从氯胺酮麻醉（100mgkg-1，IP）的痉挛性和野生型小鼠（平均年龄 P27 和 P29，分别为两性）的腰椎脊髓切片中获得。痉挛性小鼠的 GlyR 介导的 mIPSC 幅度和衰减时间常数分别降低了 25%和 50%（42.0±3.6pA 对 31.0±1.8pA，P<0.05 和 7.4±0.5ms 对 5.0±0.4ms，P<0.05；均值±SEM，n=34 和 31，分别）。对 mIPSC 幅度与上升时间和衰减时间关系的研究表明，这些差异不是由于电紧张作用引起的。GABAA 介导的 mIPSC 和 A 型钾电流的分析表明，与以前在另一种 GlyR 突变体痉挛性中观察到的突触或内在同型稳态可塑性不同，GlyR 介导的神经传递没有发生改变。应用甘氨酸到从 SDH 神经元分离的外面向外斑片中，发现痉挛性 GlyRs 的甘氨酸敏感性降低了两倍以上（EC50=130±20μM 对 64±11μM，分别；n=8 和 15，分别）。随后，使用不同的激动剂敏感性和 mIPSC 衰减时间来探测 SDH 神经元中是否存在含有 α1 的 GlyRs。基于对 1-3μM 甘氨酸的反应，甘氨酸敏感性在>75%的测试神经元中降低，并且在痉挛性样本中的衰减时间更快。总之，我们的数据表明，SDH 中的大多数 GlyRs 和甘氨酸能突触含有 α1 亚基，而很少仅由 α3 亚基组成。因此，未来设计针对 α3 亚基的治疗方法的努力必须考虑 GlyR 中 α1 和 α3 亚基之间的潜在相互作用。
