Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo III
Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo III
复制标题
使用偶氮胂 III 测量电压钳下培养的小鼠脊髓神经元中激动剂和电压门控的钙进入
作者：
ML Mayer;AB MacDermott;G. Westbrook;Sj Smith;J. Barker
采用全细胞膜片钳技术，以钙离子指示剂偶氮胂Ⅲ负载分离培养的脊髓神经元。在电压钳下，去极化电压阶跃引起钙-偶氮胂III络合物在660 nm处吸光度的瞬时增加，在570 nm处无变化。光学响应发生在阈值去极化至-30 mV时，在+10 mV时达到峰值，并随着进一步去极化而降低，这与通过电压依赖性钙通道的Ca 2+通量导致的细胞质游离钙升高一致。兴奋性氨基酸N-甲基-D-天冬氨酸（NMDA）和L-谷氨酸的内向电流反应也伴随着钙瞬变;这些是剂量依赖性的，变化与内向电流的驱动力，并被细胞外Mg 2+以电压依赖性的方式阻断，表明通过NMDA受体通道的Ca 2+通量。红藻氨酸，使君子酸，GABA的反应并不伴随着可比的钙瞬变。[Ca2由去极化电压阶跃诱发的+]i瞬变在记录开始时具有最大幅度，并随时间下降，反映了电压依赖性钙通道的衰减。相反，在持续1-2小时的全细胞记录期间，NMDA诱发的[Ca ~（2+）]i瞬变幅度逐渐增加，用Ca ~（2+）加载神经元的程序加速了NMDA诱发的[Ca ~（2+）]i瞬变幅度的增加，但减慢了电压阶跃诱发的[Ca ~（2+）]i瞬变的衰减。我们的研究结果为脊椎动物神经元跨膜钙流的两个独立来源提供了证据，即通过电压门控钙通道和通过NMDA受体通道。NMDA受体特异性激动剂门控的Ca 2+通量可能在突触可塑性、调节兴奋性和对兴奋性氨基酸的兴奋性毒性反应中发挥作用。
Spinal cord neurons is dissociated cell culture were loaded with the calcium indicator arsenazo III using the whole-cell patch-clamp recording technique. Under voltage-clamp, depolarizing voltage steps evoked transient increases in absorbance at 660 nm, with no change at 570 nm, the isosbestic wavelength for calcium-arsenazo III complexes. The optical response occurred with a threshold depolarization to -30 mV, peaked at +10 mV, and decreased with further depolarization, consistent with an elevation of cytoplasmic free calcium resulting from Ca2+ flux through voltage-dependent calcium channels. Inward current responses to the excitatory amino acids N-methyl-D-aspartic acid (NMDA) and L-glutamate were also accompanied by calcium transients; these were dose-dependent, varied with the driving force for inward current, and were blocked by extracellular Mg2+ in a voltage-dependent manner, suggesting Ca2+ flux through NMDA-receptor channels. Responses to kainate, quisqualate, and GABA were not accompanied by comparable calcium transients. [Ca2+]i transients evoked by depolarizing voltage steps were of maximal amplitude at the start of recording and declined with time, reflecting rundown of voltage-dependent calcium channels. In contrast, [Ca2+]i transients evoked by NMDA gradually increased in amplitude during periods of whole-cell recording lasting 1-2 hr. Procedures resulting in loading of the neuron with Ca2+ accelerated the increase in amplitude of [Ca2+]i transients evoked by NMDA, but slowed the decay of [Ca2+]i transients evoked by voltage steps. Our results provide evidence for 2 independent sources of transmembrane Ca2+ flux in vertebrate neurons, through voltage-gated calcium channels and through NMDA-receptor channels. The Ca2+ flux gated by NMDA-receptor- specific agonists may play a role in synaptic plasticity, in regulating excitability, and in the excitotoxic response to excitatory amino acids.
影响因子：
2.5
作者：
BROWN, TH;JOHNSTON, D
通讯作者：
JOHNSTON, D