Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland.
Drug Discovery Network Zurich, University of Zurich and ETH Zurich, Zurich, Switzerland.
J Physiol. 2023 Sep;601(18):4121-4133. doi: 10.1113/JP284589. Epub 2023 Aug 20.
Glycine receptors (GlyRs), together with GABA receptors, mediate postsynaptic inhibition in most spinal cord and hindbrain neurons. In several CNS regions, GlyRs are also expressed in presynaptic terminals. Here, we analysed the effects of a phospho-deficient mutation (S346A) in GlyR α3 subunits on inhibitory synaptic transmission in superficial spinal dorsal horn neurons, where this subunit is abundantly expressed. Unexpectedly, we found that not only were the amplitudes of evoked glycinergic inhibitory postsynaptic currents (IPSCs) significantly larger in GlyRα3(S346A) mice than in mice expressing wild-type α3GlyRs (GlyRα3(WT) mice), but so were those of GABAergic IPSCs. Decreased frequencies of spontaneously occurring glycinergic and GABAergic miniature IPSCs (mIPSCs) with no accompanying change in mIPSC amplitudes suggested a change in presynaptic transmitter release. Paired-pulse experiments on glycinergic IPSCs revealed an increased paired-pulse ratio and a smaller coefficient of variation in GlyRα3(S346A) mice, which together indicate a reduction in transmitter release probability and an increase in the number of releasable vesicles. Paired-pulse ratios of GABAergic IPSCs recorded in the presence of strychnine were not different between genotypes, while the coefficient of variation was smaller in GlyRα3(S346A) mice, demonstrating that the decrease in release probability was readily reversible by GlyR blockade, while the difference in the size of the pool of releasable vesicles remained. Taken together, our results suggest that presynaptic α3 GlyRs regulate synaptic glycine and GABA release in superficial dorsal horn neurons, and that this effect is potentially regulated by their phosphorylation status. KEY POINTS: A serine-to-alanine point mutation was introduced into the glycine receptor α3 subunit of mice. This point mutation renders α3 glycine receptors resistant to protein kinase A mediated phosphorylation but has otherwise only small effects on receptor function. Patch-clamp recordings from neurons in mouse spinal cord slices revealed an unexpected increase in the amplitudes of both glycinergic and GABAergic evoked inhibitory postsynaptic currents (IPSCs). Miniature IPSCs, paired-pulse ratios and synaptic variation analyses indicate a change in synaptic glycine and GABA release. The results strongly suggest that α3 subunit-containing glycine receptors are expressed on presynaptic terminals of inhibitory dorsal horn neurons where they regulate transmitter release.
甘氨酸受体（GlyRs）与 GABA 受体一起，介导大多数脊髓和后脑神经元的突触后抑制。在一些中枢神经系统区域，GlyRs 也在前突触末端表达。在这里，我们分析了 GlyRα3 亚基中磷酸化缺陷突变（S346A）对富含该亚基的浅层脊髓背角神经元抑制性突触传递的影响。出乎意料的是，我们发现不仅 GlyRα3（S346A）小鼠中诱发的甘氨酸能抑制性突触后电流（IPSCs）的幅度明显大于表达野生型α3GlyRs（GlyRα3（WT）小鼠）的小鼠，而且 GABA 能 IPSC 的幅度也是如此。自发发生的甘氨酸能和 GABA 能微小 IPSC（mIPSC）频率降低，而 mIPSC 幅度没有伴随变化，提示突触前递质释放发生变化。甘氨酸能 IPSC 的成对脉冲实验显示，GlyRα3（S346A）小鼠的成对脉冲比增加，变异性系数减小，这共同表明递质释放概率降低，可释放囊泡数量增加。在斯特里克林存在的情况下记录到的 GABA 能 IPSC 的成对脉冲比在两种基因型之间没有差异，而 GlyRα3（S346A）小鼠的变异性系数较小，表明 GlyR 阻断后释放概率的降低是可逆的，而可释放囊泡池的大小差异仍然存在。总之，我们的结果表明，浅层背角神经元的突触前α3 GlyRs 调节突触甘氨酸和 GABA 的释放，这种作用可能受其磷酸化状态的调节。
在小鼠甘氨酸受体α3 亚基中引入了一个丝氨酸到丙氨酸的点突变。该点突变使 α3 甘氨酸受体对蛋白激酶 A 介导的磷酸化具有抗性，但对受体功能只有很小的影响。来自小鼠脊髓切片神经元的膜片钳记录显示，甘氨酸能和 GABA 能诱发的抑制性突触后电流（IPSCs）的幅度都出乎意料地增加。微小 IPSC、成对脉冲比和突触变异性分析表明，突触甘氨酸和 GABA 的释放发生了变化。结果强烈表明，α3 亚基包含的甘氨酸受体在前抑制性背角神经元的突触前末端表达，在那里它们调节递质释放。
