Neuronal acid-induced [Zn²⁺]i elevations calibrated using the low-affinity ratiometric probe FuraZin-1.
Neuronal acid-induced [Zn²⁺]i elevations calibrated using the low-affinity ratiometric probe FuraZin-1.
复制标题
使用低亲和力比率探针 FuraZin-1 校准神经元酸诱导的 [Zn2α]i 升高。
DOI：
10.1111/jnc.13282
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发表时间：
2015
影响因子：
4.7
通讯作者：
Kiedrowski,Lech
中科院分区：
文献类型：
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作者：
Kiedrowski,Lech
这些实验是在小鼠皮质神经元的原代培养物上进行的，这些神经元来自胚胎第 16 天胎儿的冷冻保存制剂。为了校准酸诱导的细胞内 [Zn2+] ([Zn2+]i) 升高，使用了低亲和力（Kd= 39 μM，pH 6.1）比例 Zn2+ 探针 FuraZin-1。 pHi 从 7.2 下降到 6.1 导致 [Zn2+] 升高达到 2 μM；当随后使用硫醇反应剂 N-乙基马来酰亚胺 (NEM) 时，[Zn2+]i 进一步增加至 5.6 μM；使用高亲和力 Zn2+ 探针 FluoZin-3 也可以检测到类似的酸和 NEM 诱导的 [Zn2+] 升高，但无需校准。数据表明，NEM 导致 Zn2+ 从在 pH 6.1 螯合 Zn2+ 的配体中释放。 ATP 在 pH 6.1 时也可以螯合 Zn2+，因为其 pKa 约为 6.8。因此，测试了 ATP 消耗是否会影响酸诱导的 [Zn2+] 升高。通过抑制线粒体和糖酵解 ATP 的产生来诱导 ATP 消耗。有趣的是，几乎完全的 ATP 耗尽（使用荧光素/荧光素酶测定证实）未能影响酸诱导的 [Zn2+]i 增加。这些数据表明，与酸敏感的细胞内配体中积累的 Zn2+ 量相比，细胞内 ATP 依赖性储存库（Zn2+-ATP 复合物和以 ATP 依赖性方式积累 Zn2+ 的细胞器）中积累的 Zn2+ 总量可以忽略不计。在体外，酸化后，Zn2+-半胱氨酸复合物释放 Zn2+，ATP 螯合释放的 Zn2+。然而，在体内（培养的神经元）中，ATP 消耗未能增强酸诱导的 [Zn2+] 升高。这些 [Zn2+] 升高使用低亲和力比率探针 FuraZin-1 进行校准；当硫醇反应剂 N-乙基马来酰亚胺破坏半胱氨酸与 Zn2+ 的结合时，它们达到 2 µM 水平并增加至 5 µM。
The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic‐day‐16 fetuses. To calibrate acid‐induced intracelluar [Zn2+] ([Zn2+]i) elevations, a low affinity (Kd= 39 μM at pH 6.1) ratiometric Zn2+probe, FuraZin‐1, was used. A pHidrop from 7.2 to 6.1 caused [Zn2+]ielevations reaching 2 μM; when the thiol‐reactive agentN‐ethylmaleimide (NEM) was subsequently applied, [Zn2+]iincreased further to 5.6 μM; analogous acid‐ and NEM‐induced [Zn2+]ielevations could also be detected but not calibrated, using the high affinity Zn2+probe FluoZin‐3. The data indicate that NEM causes Zn2+release from ligands that chelate Zn2+at pH 6.1. ATP could also chelate Zn2+at pH 6.1 because its pKais about 6.8. Therefore, it was tested whether an ATP depletion affects the acid‐induced [Zn2+]ielevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid‐induced [Zn2+]iincreases. These data suggest that the total amount of Zn2+accumulated in intracellular ATP‐dependent stores (Zn2+‐ATP complexes and organelles that accumulate Zn2+in an ATP‐dependent manner) is negligible compared to the amount of Zn2+accumulated in the acid‐sensitive intracellular ligands.In vitro,upon acidification, Zn2+‐cysteine complexes release Zn2+and ATP chelates the released Zn2+. However,in vivo(cultured neurons), an ATP depletion failed to enhance acid‐induced [Zn2+]ielevations. These [Zn2+]ielevations were calibrated using a low affinity ratiometric probe FuraZin‐1; they reached 2 µM levels and increased to 5 µM when a thiol‐reactive agent,N‐ethylmaleimide, compromised Zn2+binding by cysteines.