Mayer M L, MacDermott A B, Westbrook G L, Smith S J, Barker J L
Laboratory of Developmental Neurobiology, NICHD, Bethesda, Maryland 20892.
J Neurosci. 1987 Oct;7(10):3230-44. doi: 10.1523/JNEUROSCI.07-10-03230.1987.
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.
使用全细胞膜片钳记录技术，将脊髓神经元解离细胞培养物用钙指示剂偶氮胂III进行负载。在电压钳制下，去极化电压阶跃引起660 nm处吸光度的瞬时增加，而在570 nm（钙-偶氮胂III复合物的等吸收波长）处无变化。光学反应在去极化至-30 mV时出现阈值，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Ca2+通量导致细胞质游离钙升高一致。对兴奋性氨基酸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；这些反应是剂量依赖性的，随内向电流的驱动力而变化，并被细胞外Mg2+以电压依赖性方式阻断，表明Ca2+通过NMDA受体通道的通量。对海人酸、quisqualate和GABA的反应未伴随着可比的钙瞬变。去极化电压阶跃引起的[Ca2+]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引起的[Ca2+]i瞬变幅度逐渐增加。导致神经元负载Ca2+的程序加速了NMDA引起的[Ca2+]i瞬变幅度的增加，但减缓了电压阶跃引起的[Ca2+]i瞬变的衰减。我们的结果为脊椎动物神经元中跨膜Ca2+通量的2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。由NMDA受体特异性激动剂门控的Ca2+通量可能在突触可塑性、调节兴奋性以及对兴奋性氨基酸的兴奋性毒性反应中起作用。
