Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607.
Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL 60612.
eNeuro. 2019 Oct 17;6(5). doi: 10.1523/ENEURO.0343-19.2019. Print 2019 Sep/Oct.
Presynaptic Ca evokes exocytosis, endocytosis, and synaptic plasticity. However, Ca flux and interactions at presynaptic molecular targets are difficult to quantify because fluorescence imaging has limited resolution. In rats of either sex, we measured single varicosity presynaptic Ca using Ca dyes as buffers, and constructed models of Ca dispersal. Action potentials evoked Ca transients with little variation when measured with low-affinity dye (peak amplitude 789 ± 39 nM, within 2 ms of stimulation; decay times, 119 ± 10 ms). Endogenous Ca buffering capacity, action potential-evoked free [Ca], and total Ca amounts entering terminals were determined using Ca dyes as buffers. These data constrained Monte Carlo (MCell) simulations of Ca entry, buffering, and removal. Simulations of experimentally-determined Ca fluxes, buffered by simulated calbindin well fit data, and were consistent with clustered Ca entry followed within 4 ms by diffusion throughout the varicosity. Repetitive stimulation caused free varicosity Ca to sum. However, simulated in nanometer domains, its removal by pumps and buffering was negligible, while local diffusion dominated. Thus, Ca within tens of nanometers of entry, did not accumulate. A model of synaptotagmin1 (syt1)-Ca binding indicates that even with 10 µM free varicosity evoked Ca, syt1 must be within tens of nanometers of channels to ensure occupation of all its Ca-binding sites. Repetitive stimulation, evoking short-term synaptic enhancement, does not modify probabilities of Ca fully occupying syt1's C2 domains, suggesting that enhancement is not mediated by Ca-syt1 interactions. We conclude that at spatiotemporal scales of fusion machines, Ca necessary for their activation is diffusion dominated.
突触前 Ca 引发胞吐、胞吞和突触可塑性。然而，由于荧光成像的分辨率有限，突触前分子靶点的 Ca 流和相互作用难以量化。在雄性和雌性大鼠中，我们使用 Ca 染料作为缓冲液测量单个囊泡突触前 Ca，并构建 Ca 弥散模型。使用低亲和力染料测量时，动作电位引发的 Ca 瞬变变化很小（峰值幅度 789±39 nM，刺激后 2 ms 内；衰减时间 119±10 ms）。使用 Ca 染料作为缓冲液确定内源性 Ca 缓冲能力、动作电位引发的游离[Ca]和进入末梢的总 Ca 量。这些数据约束了 Ca 进入、缓冲和去除的蒙特卡罗（MCell）模拟。模拟实验确定的 Ca 通量，用模拟钙结合蛋白缓冲，很好地符合数据，并且与簇状 Ca 进入一致，随后在 4 ms 内扩散到整个囊泡。重复刺激导致游离囊泡 Ca 累积。然而，在纳米域中模拟时，泵和缓冲对其去除可忽略不计，而局部扩散占主导地位。因此，进入数十纳米范围内的 Ca 不会累积。突触融合蛋白 1（syt1）-Ca 结合的模型表明，即使在 10 µM 游离囊泡引发的 Ca 条件下，syt1 也必须在通道的数十纳米范围内，以确保其所有 Ca 结合位点都被占据。重复刺激，引发短期突触增强，不会改变 Ca 完全占据 syt1 的 C2 结构域的概率，表明增强不是由 Ca-syt1 相互作用介导的。我们得出结论，在融合机器的时空尺度上，激活它们所需的 Ca 由扩散主导。
