Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals
Mitochondrial Free Ca2+ Levels and Their Effects on Energy Metabolism in Drosophila Motor Nerve Terminals
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DOI：
10.1016/j.bpj.2013.03.064
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发表时间：
2013-06-04
影响因子：
3.4
通讯作者：
Macleod, Gregory T.
中科院分区：
文献类型：
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作者：
Ivannikov, Maxim V.;Macleod, Gregory T.
线粒体Ca ~（2+）摄取对线粒体有双重作用。Ca 2+在线粒体基质中的积累消耗膜电位（（Delta）Psi（m）），但线粒体内酶的Ca 2+结合加速氧化磷酸化，导致线粒体超极化。尚未确定在神经末梢中的线粒体中触发这些代谢反应的基质游离Ca 2+（[Ca 2 +]（m））的水平。在这里，我们估计[Ca 2 +]（m）在运动神经元末梢的果蝇幼虫使用两种方法：两种化学Ca 2+指标的相对反应与20倍的差异，在Ca 2+亲和力（rhod-FF和rhod-5 N），和响应低亲和力，遗传编码的比率Ca 2+指标（D4 cpv）校准对已知的Ca 2+水平。基质pH值（pH值（m））和（Δ）Psi（m）分别使用比率pericam和四甲基罗丹明乙酯探针，以确定何时线粒体能量代谢升高。在休息时，[Ca 2 +]（m）为0.22 +/- 0.04 μ M，但当轴突发射接近其内源频率仅2秒时，[Ca 2 +]（M）上升至接近26 μ M（rhod-FF/rhod-5 N为24.3 +/- 3.4 μ M，D4 cpv为27.0 +/- 2.6 μ M）。这种[Ca（2+）] m的升高与pH（m）的快速升高相一致，随后是刺激后（Delta）Psi（m）超极化。然而，pHm降低，并且没有观察到响应于较低水平的[Ca 2 +]（m）（高达13.1 μ M）的（Delta）Psi（m）超极化。这些数据表明，令人惊讶的高水平的[Ca 2 +]（m）需要刺激突触前线粒体能量代谢。
Mitochondrial Ca2+ uptake exerts dual effects on mitochondria. Ca2+ accumulation in the mitochondrial matrix dissipates membrane potential ((Delta)Psi(m)), but Ca2+ binding of the intramitochondrial enzymes accelerates oxidative phosphorylation, leading to mitochondrial hyperpolarization. The levels of matrix free Ca2+ ([Ca2+](m)) that trigger these metabolic responses in mitochondria in nerve terminals have not been determined. Here, we estimated [Ca2+](m) in motor neuron terminals of Drosophila larvae using two methods: the relative responses of two chemical Ca2+ indicators with a 20-fold difference in Ca2+ affinity (rhod-FF and rhod-5N), and the response of a low-affinity, genetically encoded ratiometric Ca2+ indicator (D4cpv) calibrated against known Ca2+ levels. Matrix pH (pH(m)) and (Delta)Psi(m) were monitored using ratiometric pericam and tetramethylrhodamine ethyl ester probe, respectively, to determine when mitochondrial energy metabolism was elevated. At rest, [Ca2+](m) was 0.22 +/- 0.04 mu M, but it rose to similar to 26 mu M (24.3 +/- 3.4 mu M with rhod-FF/rhod-5N and 27.0 +/- 2.6 mu M with D4cpv) when the axon fired close to its endogenous frequency for only 2 s. This elevation in [Ca(2+])m coincided with a rapid elevation in pH(m) and was followed by an after-stimulus (Delta)Psi(m) hyperpolarization. However, pHm decreased and no (Delta)Psi(m) hyperpolarization was observed in response to lower levels of [Ca2+](m), up to 13.1 mu M. These data indicate that surprisingly high levels of [Ca2+](m) are required to stimulate presynaptic mitochondrial energy metabolism.