Sobolevsky A I, Koshelev S G, Khodorov B I
Institute of General Pathology and Pathophysiology, 125315 Moscow, Russia.
J Neurosci. 1999 Dec 15;19(24):10611-26. doi: 10.1523/JNEUROSCI.19-24-10611.1999.
Using whole-cell patch-clamp techniques, we studied the interaction of open NMDA channels with tetraalkylammonium compounds: tetraethylammonium (TEA), tetrapropylammonium (TPA), tetrabutylammonium (TBA), and tetrapentylammonium (TPentA). Analysis of the blocking kinetics, concentration, and agonist dependencies using a set of kinetic models allowed us to create the criteria distinguishing the effects of these blockers on the channel closure, desensitization, and agonist dissociation. Thus, it was found that TPentA prohibited, TBA partly prevented, and TPA and TEA did not prevent either the channel closure or the agonist dissociation. TPentA and TBA prohibited, TPA slightly prevented, and TEA did not affect the channel desensitization. These data along with the voltage dependence of the stationary current inhibition led us to hypothesize that: (1) there are activation and desensitization gates in the NMDA channel; (2) these gates are distinct structures located in the external channel vestibule, the desensitization gate being located deeper than the activation gate. The size of the blocker plays a key role in its interaction with the NMDA channel gating machinery: small blockers (TEA and TPA) bind in the depth of the channel pore and permit the closure of both gates, whereas larger blockers (TBA) allow the closure of the activation gate but prohibit the closure of the desensitization gate; finally, the largest blockers (TPentA) prohibit the closure of both activation and desensitization gates. The mean diameter of the NMDA channel pore in the region of the activation gate localization was estimated to be approximately 11 A.
我们使用全细胞膜片钳技术，研究了开放的N-甲基-D-天冬氨酸（NMDA）通道与四烷基铵化合物之间的相互作用：四乙铵（TEA）、四丙铵（TPA）、四丁铵（TBA）和四戊铵（TPentA）。使用一组动力学模型分析阻断动力学、浓度和激动剂依赖性，使我们能够建立区分这些阻断剂对通道关闭、脱敏和激动剂解离影响的标准。因此，发现TPentA可阻止，TBA部分阻止，而TPA和TEA既不能阻止通道关闭也不能阻止激动剂解离。TPentA和TBA可阻止，TPA轻微阻止，而TEA不影响通道脱敏。这些数据以及稳态电流抑制的电压依赖性使我们推测：（1）NMDA通道中存在激活门和脱敏门；（2）这些门是位于通道外部前庭的不同结构，脱敏门比激活门更深。阻断剂的大小在其与NMDA通道门控机制的相互作用中起关键作用：小的阻断剂（TEA和TPA）结合在通道孔的深处，允许两个门关闭，而较大的阻断剂（TBA）允许激活门关闭但阻止脱敏门关闭；最后，最大的阻断剂（TPentA）阻止激活门和脱敏门关闭。估计激活门定位区域中NMDA通道孔的平均直径约为11埃。
