Legendre P, Westbrook G L
Vollum Institute, Oregon Health Sciences University, Portland 97201.
Mol Pharmacol. 1991 Aug;40(2):289-98.
The inhibition of N-methyl-D-aspartate (NMDA) receptor channels by the vasodilatory and anti-ischemic agent ifenprodil was examined on cultured rat hippocampal neurons. Whole-cell and single-channel patch recordings were used. Ifenprodil inhibition of NMDA currents could be separated into two components, with IC50 values of 0.75 and 161 microM. The high and low affinity components were both voltage independent but could be separated by their kinetics and dependence on extracellular calcium and glycine. The maximal inhibition of inward current by ifenprodil (approximately 90%) was equally divided between the two components in 0.3 mM extracellular calcium and 500 nM glycine. The low affinity action of ifenprodil had rapid kinetics and appeared to result from allosteric inhibition of the glycine modulatory site on the NMDA receptor. The macroscopic kinetics of the high affinity component were slow. The rate of onset was concentration dependent, and complete recovery required 1-2 min. Unlike open-channel blockers, ifenprodil block was not use dependent, and pre-exposure to ifenprodil also reduced subsequent NMDA responses. Low concentrations of ifenprodil were less effective after calcium-dependent inactivation of whole-cell currents, but the IC50 was unaffected, suggesting that calcium and ifenprodil act on a common set of channels. On outside-out membrane patches, ifenprodil reduced the frequency of channel opening without altering the single-channel conductance. Open time histograms of the large conductance events revealed two mean open times of approximately 2 and 8 msec, but only the duration of the long openings was decreased by ifenprodil. This effect was concentration dependent and revealed a blocking rate constant of 6 x 10(7) M-1sec-1. However, the proportion of current blocked by low concentrations of ifenprodil was larger in outside-out patches than in whole-cell recordings, suggesting that intracellular factors may influence ifenprodil efficacy. These results indicate that high affinity ifenprodil binding is extracellular and does not require agonist binding or channel opening. Because low concentrations of ifenprodil only partially inhibited the current and affected only the long openings, ifenprodil may promote a modal shift in channel gating.
在培养的大鼠海马神经元上研究了血管舒张和抗缺血药物艾芬地尔对N-甲基-D-天冬氨酸（NMDA）受体通道的抑制作用。采用全细胞和单通道膜片钳记录。艾芬地尔对NMDA电流的抑制可分为两个成分，IC50值分别为0.75和161μM。高亲和力和低亲和力成分均与电压无关，但可根据其动力学以及对细胞外钙和甘氨酸的依赖性进行区分。在0.3 mM细胞外钙和500 nM甘氨酸条件下，艾芬地尔对内向电流的最大抑制（约90%）在两个成分之间平均分配。艾芬地尔的低亲和力作用具有快速动力学，似乎是由对NMDA受体上甘氨酸调节位点的变构抑制引起的。高亲和力成分的宏观动力学较慢。起效速率与浓度有关，完全恢复需要1 - 2分钟。与开放通道阻滞剂不同，艾芬地尔的阻断不具有使用依赖性，预先暴露于艾芬地尔也会降低随后的NMDA反应。全细胞电流钙依赖性失活后，低浓度的艾芬地尔效果较差，但IC50不受影响，这表明钙和艾芬地尔作用于同一组通道。在外侧向外的膜片上，艾芬地尔降低了通道开放频率，而不改变单通道电导。大电导事件的开放时间直方图显示出两个平均开放时间，分别约为2和8毫秒，但只有长开放时间的持续时间被艾芬地尔缩短。这种效应与浓度有关，显示出阻断速率常数为6×10⁷ M⁻¹秒⁻¹。然而，外侧向外膜片中低浓度艾芬地尔阻断的电流比例大于全细胞记录中的比例，这表明细胞内因素可能影响艾芬地尔的疗效。这些结果表明，高亲和力的艾芬地尔结合在细胞外，不需要激动剂结合或通道开放。由于低浓度的艾芬地尔仅部分抑制电流且仅影响长开放时间，艾芬地尔可能促进通道门控的模式转变。
