Probing of NMDA channels with fast blockers
Probing of NMDA channels with fast blockers
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DOI：
10.1523/jneurosci.19-24-10611.1999
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发表时间：
1999-12-15
影响因子：
5.3
通讯作者：
Khodorov, BI
中科院分区：
文献类型：
--
作者：
Sobolevsky, AI;Koshelev, SG;Khodorov, BI
利用全细胞膜片钳技术，我们研究了开放的NMDA通道与四烷基铵化合物的相互作用：四乙基铵（TEA）、四丙基铵（TPA）、四丁基铵（TBA）和四戊基铵（TPentA）。使用一组动力学模型对阻断动力学、浓度和激动剂依赖性进行分析，使我们能够创建区分这些阻断剂对通道关闭、脱敏和激动剂解离的影响的标准。因此，发现TPentA禁止，TBA部分阻止，TPA和TEA既不阻止通道关闭也不阻止激动剂解离。TPentA和TBA禁用，TPA略有阻止，TEA不影响通道脱敏。这些数据以及稳定电流抑制的电压依赖性使我们假设：(1)在NMDA通道中存在激活和脱敏门；(2)这些门是位于外通道前庭的独特结构，脱敏门的位置比激活门深。阻滞剂的大小在其与NMDA通道门机制的相互作用中起着关键作用：小阻滞剂（TEA和TPA）结合在通道孔的深处，并允许关闭两个门，而较大的阻滞剂（TBA）允许关闭激活门，但禁止关闭脱敏门；最后，最大的阻滞剂（TPentA）禁止激活和脱敏门的关闭。在激活栅定位区域，NMDA通道孔的平均直径约为11埃。
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 similar to 11 Angstrom.