Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3EG, United Kingdom.
Synapse. 2013 Nov;67(11):729-40. doi: 10.1002/syn.21678. Epub 2013 Jun 3.
Both intracellular pH (pHi) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH- and Ca(2+) -sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca(2+) i rises and pHi falls (∼0.1 pH units) followed by recovery of both Ca(2+) i and pHi. The plasma-membrane calcium ATPase (PMCA) inhibitor, 5(6)-carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium-independent role for the pHi shifts in modulating vesicle fusion we recorded post-synaptic miniature end-plate potential (mEPP) and current (mEPC) frequency in Ca(2+) -free solution. Acidification by propionate superfusion, NH(4)(+) withdrawal, or the inhibition of acid extrusion on the Na(+)/H(+) exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH(4)(+) removal. In the presence of NH(4)(+), 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca(2+) -dependent pHi shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering.
神经元活动期间细胞内 pH 值 (pHi) 和突触间隙 pH 值都会发生变化，但对于这些 pH 值变化如何通过影响囊泡融合来影响突触传递知之甚少。为了解决这个问题，我们在神经肌肉接头的末梢中成像了 pH 值和 Ca(2+) 敏感的荧光指示剂（HPTS，Oregon green）。刺激运动神经会引发突触前 Ca(2+) i 上升和 pHi 下降（约 0.1 pH 单位），随后 Ca(2+) i 和 pHi 都恢复。质膜钙 ATP 酶（PMCA）抑制剂 5(6)-羧基荧光素二乙酸酯，使钙恢复和酸化都减缓。为了研究 pH 值变化在调节囊泡融合中的可能的钙非依赖性作用，我们在无 Ca(2+) 溶液中记录了突触后微小终板电位（mEPP）和电流（mEPC）频率。丙酸盐超灌注、NH(4)(+) 耗尽或 Na(+)/H(+) 交换器（NHE）的酸外排抑制会引起微小频率升高。此外，酸外排抑制增强了丙酸盐添加和 NH(4)(+) 去除引起的上升。在 NH(4)(+) 存在的情况下，23 个细胞中有 10 个在延迟后显示微小频率的一次或多次上升。这些发现表明，由 PMCA 引起并由 NHE 调节的 Ca(2+) 依赖性 pHi 变化可能会刺激囊泡释放。此外，在存在膜通透缓冲剂的情况下，胞吐的酸或其等效物可能通过正反馈增强释放。这种迄今为止被忽视的 pH 信号，以及囊泡酸的潜在反馈作用，可以解释与 pH 值变化及其缓冲相关的一些重要神经元兴奋性变化。
