Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
Neurobiology Department, MRC Laboratory of Molecular Biology Cambridge Biomedical Campus Francis Crick Avenue, Cambridge, UK.
J Physiol. 2022 Jan;600(2):333-347. doi: 10.1113/JP282171. Epub 2021 Dec 8.
Many pentameric ligand-gated ion channels are modulated by extracellular pH. Glycine receptors (GlyRs) share this property, but it is not well understood how they are affected by pH changes. Whole cell experiments on HEK293 cells expressing zebrafish homomeric α1 GlyR confirmed previous reports that acidic pH (6.4) reduces GlyR sensitivity to glycine, whereas alkaline pH (8.4) has small or negligible effects. In addition to that, at pH 6.4 we observed a reduction in the maximum responses to the partial agonists β-alanine and taurine relative to the full agonist glycine. In cell-attached single-channel recording, low pH reduced agonist efficacy, as the maximum open probability decreased from 0.97, 0.91 and 0.66 to 0.93, 0.57 and 0.34 for glycine, β-alanine and taurine, respectively, reflecting a threefold decrease in efficacy equilibrium constants for all three agonists. We also tested the effect of pH 6.4 in conditions that replicate those at the native synapse, recording outside-out currents elicited by fast application of millisecond pulses of agonists on α1 and α1β GlyR, at a range of intracellular chloride concentrations. Acidic pH reduced the area under the curve of the currents, by reducing peak amplitude, slowing activation and speeding deactivation. Our results show that acidification of the extracellular pH by one unit, as may occur in pathological conditions such as ischaemia, impairs GlyR gating and is likely to reduce the effectiveness of glycinergic synaptic inhibition. KEY POINTS: Extracellular pH in the central nervous system (CNS) is known to shift towards acidic values during pathophysiological conditions such as ischaemia and seizures. Acidic extracellular pH is known to affect GABAergic inhibitory synapses, but its effect on signals mediated by glycine receptors (GlyR) is not well characterised. Moderate acidic conditions (pH 6.4) reduce the maximum single channel open probability of recombinant homomeric GlyRs produced by the neurotransmitter glycine or other agonists, such as β-alanine and taurine. When glycine was applied with a piezoelectric stepper to outside out patches, to simulate its fast rise and short duration at the synapse, responses became shorter and smaller at pH 6.4. The effect was also observed with physiologically low intracellular chloride and in mammalian heteromeric GlyRs. This suggests that acidic pH is likely to reduce the strength of inhibitory signalling at glycinergic synapses.
许多五聚体配体门控离子通道受细胞外 pH 值调节。甘氨酸受体（GlyRs）具有这种特性，但尚不清楚它们如何受到 pH 值变化的影响。在表达斑马鱼同源α1 GlyR 的 HEK293 细胞上进行的全细胞实验证实了先前的报告，即酸性 pH（6.4）降低了 GlyR 对甘氨酸的敏感性，而碱性 pH（8.4）的影响较小或可以忽略不计。除此之外，在 pH 6.4 时，我们观察到与完全激动剂甘氨酸相比，对部分激动剂β-丙氨酸和牛磺酸的最大反应减少。在细胞贴附的单通道记录中，低 pH 值降低了激动剂的效能，因为最大开放概率从 0.97、0.91 和 0.66 分别降低到 0.93、0.57 和 0.34，反映了所有三种激动剂的效能平衡常数降低了三倍。我们还测试了 pH 6.4 在复制天然突触条件下的效果，通过快速施加毫秒脉冲激动剂，在一系列细胞内氯离子浓度下记录α1 和α1β GlyR 引发的外向电流。酸性 pH 值通过降低峰值幅度、减缓激活和加速失活来减少电流的曲线下面积。我们的结果表明，细胞外 pH 值降低一个单位，如在缺血等病理条件下可能发生的那样，会损害 GlyR 的门控作用，并且可能降低甘氨酸能抑制性突触抑制的有效性。关键点：中枢神经系统（CNS）中的细胞外 pH 值在缺血和癫痫等病理生理条件下已知会向酸性值偏移。已知酸性细胞外 pH 值会影响 GABA 能抑制性突触，但对甘氨酸受体（GlyR）介导的信号的影响尚未很好地描述。中等酸性条件（pH 6.4）降低了由神经递质甘氨酸或其他激动剂（如β-丙氨酸和牛磺酸）产生的重组同源 GlyR 的最大单通道开放概率。当用压电步进器将甘氨酸施加到外面的斑块上时，以模拟其在突触处的快速上升和短暂持续时间，在 pH 6.4 时，反应变得更短更小。在生理低细胞内氯离子浓度和在哺乳动物异源 GlyR 中也观察到了这种情况。这表明酸性 pH 值可能会降低甘氨酸能突触抑制信号的强度。
