Zheng X, Zhang L, Wang A P, Araneda R C, Lin Y, Zukin R S, Bennett M V
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Neuroscience. 1999;93(1):125-34. doi: 10.1016/s0306-4522(99)00154-2.
Spermine and other endogenous polyamines potentiate, block and permeate the N-methyl-D-aspartate receptor channel. To identify structural determinants of the N-methyl-D-aspartate channel that mediate spermine's actions, we generated mutant receptors with asparagine (N) to glutamine (Q) or arginine (R) substitutions in the selectivity filter of the channel. We demonstrate that mutation of the three critical asparagines in this domain differentially affects block by phencyclidine and both potentiation and block by spermine. N-to-Q and N-to-R mutations in the N site of the NR1 subunit (N598 in NR1(011), N619 in NR1(100)) and N-to-Q mutations in the N and N + 1 sites (N595 and N596 in NR2A, respectively) of the NR2 subunit (Q/NN, R/NN, N/QN, N/NQ, Q/QN and Q/NQ receptors) reduced affinity for phencyclidine. The Q/NN receptor showed markedly reduced potentiation by spermine, with little or no change in spermine block. The R/NN receptor showed markedly reduced spermine potentiation and affinity for spermine at its block site. The N/QN, N/NQ and Q/QN mutant receptors showed somewhat enhanced spermine block, while the Q/ NQ double mutant exhibited significantly more enhanced spermine block. Thus, the asparagine residues critical to Ca2+ permeability and Mg2+ block of N-methyl-D-aspartate channels are also critical to block by spermine and phencyclidine. To examine the interaction of spermine and phencyclidine within the channel, we performed competition studies. Spermine appeared to compete with phencyclidine for binding to the receptor; however, blocks by phencyclidine and by spermine were not additive. The findings suggest that spermine can bind to a site in the external vestibule of the channel to impede phencyclidine binding, but allow Na+ influx.
精胺和其他内源性多胺可增强、阻断并通透N-甲基-D-天冬氨酸受体通道。为了确定介导精胺作用的N-甲基-D-天冬氨酸通道的结构决定因素，我们构建了通道选择性过滤器中天冬酰胺（N）被谷氨酰胺（Q）或精氨酸（R）取代的突变受体。我们证明，该结构域中三个关键天冬酰胺的突变对苯环利定的阻断以及精胺的增强作用和阻断作用有不同影响。NR1亚基N位点（NR1(011)中的N598、NR1(100)中的N619）的N-to-Q和N-to-R突变以及NR2亚基N和N + 1位点（NR2A中分别为N595和N596）的N-to-Q突变（Q/NN、R/NN、N/QN、N/NQ、Q/QN和Q/NQ受体）降低了对苯环利定的亲和力。Q/NN受体对精胺的增强作用明显降低，而精胺阻断作用几乎没有变化。R/NN受体对精胺的增强作用和在其阻断位点对精胺的亲和力明显降低。N/QN、N/NQ和Q/QN突变受体对精胺的阻断作用略有增强，而Q/NQ双突变体对精胺的阻断作用显著增强。因此，对N-甲基-D-天冬氨酸通道Ca2+通透性和Mg2+阻断至关重要的天冬酰胺残基对精胺和苯环利定的阻断也至关重要。为了研究精胺和苯环利定在通道内的相互作用，我们进行了竞争研究。精胺似乎与苯环利定竞争与受体的结合；然而，苯环利定和精胺的阻断作用并非相加的。这些发现表明，精胺可结合到通道外部前庭的一个位点，以阻止苯环利定结合，但允许Na+内流。
