Comparison of quantitative calcium flux through NMDA, ATP, and ACh receptor channels.
Comparison of quantitative calcium flux through NMDA, ATP, and ACh receptor channels.
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DOI：
10.1016/s0006-3495(95)80211-0
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发表时间：
1995-02
影响因子：
3.4
通讯作者：
Marc Rogers;John A. Dani
中科院分区：
文献类型：
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作者：
Marc Rogers;John A. Dani
NMDA受体、ATP受体和烟碱型ACh受体对激动剂的反应是通过构象变化打开对钙离子有通透性的弱选择性阳离子通道。我们通过使用全细胞膜片钳技术同时测量膜电流和使用荧光指示剂Fura-2测量细胞内钙离子来确定钙离子携带的电流的比例。Fura-2对游离Ca~(2+)的反应分别针对每个细胞进行了校准。比较了两种不同的校准方法：一种是使用电压激活的钙通道，另一种是在纯钙溶液中使用相同的配体门控通道。这两种方法得出的结果在数量上是不同的。该方法使用通过配基门控通道的纯钙电流来校准Fura-2信号，通过产生测试反应的相同内流途径，从而控制通道的分布，并确保传入的Ca~(2+)和Fura-2之间有类似的相互作用。在-50 mV、含2.5 mM Ca~(2+)的生理溶液中，海马神经元上钙离子通过NMDA受体的内向电流百分比为12.4%。相比之下，在交感神经元中，钙离子通过神经元nAChRs的电流百分比为4.7%，通过ATP激活的嘌呤能受体的电流百分比为6.5%。这些百分比可以用来估计突触激活期间通过这些受体进入的钙离子的量，但必须谨慎考虑每个受体的许多亚型。
NMDA receptors, ATP receptors, and nicotinic ACh receptors respond to agonist by undergoing conformational changes that open weakly selective cationic channels that are permeable to calcium. We determined the fraction of the current carried by calcium by simultaneously measuring membrane current using whole-cell patch-clamp techniques and intracellular Ca2+ using the fluorescent indicator Fura-2. The Fura-2 response to free Ca2+ was calibrated individually for each cell. Two different calibration methods are compared: one uses voltage-activated Ca2+ channels, and the other uses the same ligand-gated channels that are being tested but in a pure Ca2+ solution. The two methods give quantitatively different results. The method using pure Ca2+ currents through ligand-gated channels calibrates the Fura-2 signal through the same influx pathway that generates the test response, thus controlling for the distribution of channels and ensuring a similar interaction between the incoming Ca2+ and Fura-2. In a physiologic solution containing 2.5 mM Ca2+ at a holding potential of -50 mV, the percentage of inward current carried by Ca2+ through NMDA receptors in hippocampal neurons is 12.4%. By comparison, in sympathetic neurons the percentage of current carried by Ca2+ through neuronal nAChRs is 4.7%, and through ATP-activated purinergic receptors it is 6.5%. These percentages can be used to estimate the amount of Ca2+ entry through these receptors during synaptic activation, but care must be exercised in considering the many subtypes of each receptor.