Sites of antagonist action on N-methyl-D-aspartic acid receptors studied using fluctuation analysis and a rapid perfusion technique.
Sites of antagonist action on N-methyl-D-aspartic acid receptors studied using fluctuation analysis and a rapid perfusion technique.
复制标题
使用波动分析和快速灌注技术研究 N-甲基-D-天冬氨酸受体拮抗剂作用位点。
DOI：
10.1152/jn.1988.60.2.645
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发表时间：
1988
影响因子：
2.5
通讯作者：
Ladislav Vyklicky
中科院分区：
文献类型：
--
作者：
M. Mayer;G. Westbrook;Ladislav Vyklicky
1.分离培养的小鼠海马神经元以低密度生长在预先接种的海马神经胶质细胞培养物上，并使用紧密密封的全细胞膜片钳技术进行电压钳位。采用流管快速交换细胞外液，并应用N-甲基-D-天冬氨酸（NMDA）和一些NMDA拮抗剂。使用波动分析来估计在应用拮抗剂期间NMDA激活的离子通道的行为的变化。在NMDA的存在下，对照光谱通过与5-6 ms的平均开放时间一致的单洛伦兹函数很好地拟合。认为在NMDA受体激动剂识别位点起作用的两种拮抗剂，2-氨基-5-膦酰基戊酸（AP 5）和犬尿烯酸，没有产生平均开放时间或单通道电导的变化，这与它们作为竞争性拮抗剂的作用一致。拮抗作用的发生和从AP 5和犬尿烯酸的作用中的恢复在1秒内迅速且完全。然而，将细胞外甘氨酸浓度从1 μ M提高至1 mM，降低了100 μ M犬尿烯酸作为NMDA拮抗剂的效力，表明犬尿烯酸在NMDA受体通道上的甘氨酸调节位点处具有作为竞争性拮抗剂的额外作用。3.在存在150 μ M镁的情况下，在-60 mV下记录的NMDA光谱通过双洛伦兹函数拟合，与由持续时间为3.3 ms的开放突发组成的单通道事件一致，被平均持续时间为0.18 ms的阻断和解除阻断事件中断。镁拮抗作用的开始和恢复迅速，并在1 s内完成，但具有高度电压依赖性，在+40 mV时，镁（150 μ M）不能产生NMDA拮抗作用。这些结果与通过镁与离子通道内的位点结合产生的NMDA受体通道的电压依赖性通道阻滞一致。4.锌（30 μ M）在-60 mV和+40 mV下均为有效的NMDA拮抗剂，并且在任一电位下似乎将NMDA激活离子通道的平均开放时间从约5 ms减少至约3 ms。在所测量的频率范围内（1- 1，000 Hz），在锌拮抗作用期间，NMDA光谱与单洛伦兹曲线拟合良好，这与用镁获得的结果相反。在锌存在下，平均单通道电导也降低至对照的约75%。拮抗作用的开始和从锌的作用中的恢复在1秒内是迅速和完全的。（400字处截断摘要）
1. Mouse hippocampal neurons in dissociated culture were grown at low density on previously plated hippocampal glial cell cultures and voltage clamped using the tight seal whole-cell patch-clamp technique. Flow pipes were used to rapidly exchange the extracellular solution, and to apply N-methyl-D-aspartic acid (NMDA) and some NMDA antagonists. Fluctuation analysis was used to estimate changes in the behavior of NMDA-activated ion channels during application of antagonists. In the presence of NMDA control spectra were well fit by single Lorentzian functions consistent with mean open times of 5-6 ms. 2. Two antagonists thought to act at the NMDA receptor agonist recognition site, 2-amino-5-phosphonovaleric acid (AP5) and kynurenic acid, did not produce changes in the mean open time or single channel conductance, consistent with their action as competitive antagonists. Onset of antagonism and recovery from the action of both AP5 and kynurenic acid was rapid and complete within 1 s. However, raising the extra-cellular glycine concentration, from 1 microM to 1 mM, reduced the potency of 100 microM kynurenic acid as an NMDA antagonist, suggesting that kynurenate has an additional action as a competitive antagonist at the glycine modulatory site on NMDA receptor channels. 3. In the presence of 150 microM magnesium NMDA spectra recorded at -60 mV were fit by double Lorentzian functions, consistent with single-channel events consisting of bursts of openings lasting 3.3 ms in duration, interrupted by blocking and unblocking events of average duration 0.18 ms. The onset and recovery from magnesium antagonism was rapid, and complete within 1 s, but was highly voltage dependent and at +40 mV magnesium (150 microM) failed to produce NMDA antagonism. These results are consistent with a voltage-dependent channel block of NMDA receptor channels produced by binding of magnesium to a site within the ion channel. 4. Zinc (30 microM) was a potent NMDA antagonist at both -60 and +40 mV, and at either potential appeared to reduce the mean open time of NMDA-activated ion channels from about 5 ms to approximately 3 ms. Over the frequency range measured, 1-1,000 Hz, NMDA spectra were well fit by single Lorentzians during zinc antagonism, in contrast to results obtained with magnesium. The mean single channel conductance also decreased in the presence of zinc to approximately 75% of control. Onset of antagonism and recovery from the action of zinc was rapid and complete within 1 s.(ABSTRACT TRUNCATED AT 400 WORDS)