Pharmacological characterization of presynaptic calcium channels using subsecond biochemical measurements of synaptosomal neurosecretion.
Pharmacological characterization of presynaptic calcium channels using subsecond biochemical measurements of synaptosomal neurosecretion.
复制标题
使用突触体神经分泌的亚秒生化测量来表征突触前钙通道的药理学特征。
DOI：
10.1016/0028-3908(95)00133-q
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发表时间：
1995
影响因子：
4.7
通讯作者：
Dunlap,K
中科院分区：
文献类型：
--
作者：
Turner,TJ;Dunlap,K
选择性阻断神经元钙通道亚型的肽拮抗剂的最新发展为研究突触前钙通道在触发胞吐中的作用提供了工具。多种方法一致证明多种通道类型参与胞吐作用。我们研究了大鼠皮层突触体释放[3H]谷氨酸的亚秒动力学，作为突触前钙通道活性的测定。该系统已经在广泛的条件下进行了表征，以比较递质释放的生化测量与突触电流的电生理测量。ω-agatoxin IVA， ω-conotoxins GVIA和MVIIC在Ca2+内流降低的情况下(1)降低KCl浓度以减少去极化的程度，(2)降低Ca2+浓度，或(3)部分阻断Ca2+内流与其他拮抗剂之一。通过联合使用这些毒素，我们发现至少有三种不同的药理学通道参与胞吐作用。ω-蛇形毒素IVA （IC50= 12.2 nM）和ω-蛇形毒素MVIIC （IC50= 35 nM）阻断谷氨酸释放的比例最大，符合P型通道的药理作用。饱和浓度（1 μM）的cu-agatoxin IVA和co- concontoxin MVIIC的作用相互阻断，表明这两种肽完全重叠。特异性N型拮抗剂ω-conotoxin GVIA抑制了很大一部分的释放（ic50小于1 nM），但仅在Ca2+浓度降低的条件下。这些结果表明，神经末梢的N型通道与海马体细胞中的N型通道不同，因为它似乎对ω- concontoxin MVIIC具有抗性。当Ca2+浓度降低（0.46 mM或以下）时，ω- concontoxin GVIA （100 nM）与co-agatoxin IVA或ω- concontoxin MVIIC（各1 μM）的组合阻断了约90%的释放，但当刺激缓冲液中Ca2+浓度为1 mM或更高时，仍有30-40%的释放，表明抗性通道类型(s)也参与胞外作用。这种抗性表型的特异性抑制剂将有助于进一步完善我们对突触前钙通道在介导神经分泌中的作用的理解。
The recent development of peptide antagonists that selectively block subtypes of neuronal calcium channel has provided tools to study the role of presynaptic calcium channels in triggering exocytosis. A variety of methods have consistently demonstrated that multiple channel types participate in exocytosis. We have studied the subsecond kinetics of [3H]glutamate release from rat cortical synaptosomes as an assay for presynaptic calcium channel activity. The system has been characterized over a broad range of conditions in an effort to compare biochemical measurements of transmitter release with electrophysiological measurements of synaptic currents. The efficacies of ω-agatoxin IVA and ω-conotoxins GVIA and MVIIC were increased when Ca2+influx was decreased by: (1) decreasing the KCl concentration to diminish the extent of depolarization, (2) decreasing the Ca2+concentration, or (3) partially blocking Ca2+influx with one of the other antagonists. By using these toxins in combination, we found that at least three types of pharmacologically distinct channel participate in exocytosis. The largest fraction of glutamate release is blocked by ω-agatoxin IVA (IC50= 12.2 nM) and by ω-conotoxin MVIIC (IC50= 35 nM), consistent with the pharmacology of a P type channel. The effects of saturating concentrations (1 μM) of cu-agatoxin IVA or co-conotoxin MVIIC occlude each other, suggesting that these peptides overlap completely. The specific N type antagonist ω-conotoxin GVIA inhibits a significant portion of release (IC50less than 1 nM) but only under conditions of reduced Ca2+concentration. These results suggest that the N type channel in nerve terminals is distinct from that found in hippocampal somata, since it appears to be resistant to by ω-conotoxin MVIIC. The combination of ω-conotoxin GVIA (100 nM) and either co-agatoxin IVA or ω-conotoxin MVIIC (1 μM each) blocked approx 90% of release when the Ca2+concentration was reduced (0.46 mM or less), but 30–40% of release remained when the concentration of Ca2+in the stimulus buffer was 1 mM or greater, indicating that a resistant channel type(s) also participates in exocytosis. Specific inhibitors of this resistant phenotype will be useful for further refinement of our understanding of the role of presynaptic calcium channels in mediating neurosecretion.