Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+
Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+
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DOI：
10.1111/j.1469-7793.1998.013bx.x
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发表时间：
1998-01-01
影响因子：
5.5
通讯作者：
Sakmann, B
中科院分区：
文献类型：
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作者：
Wollmuth, LP;Kuner, T;Sakmann, B
1. 细胞外Mg2+对n -甲基-d -天冬氨酸（NMDA）受体通道的电压依赖性阻断是其对中枢神经系统突触生理学贡献的关键决定因素。通过分析位于该区域或附近的暴露残基上的一组不同氨基酸取代的影响，研究了通道狭窄在确定块中的作用。NMDA受体通道由野生型和突变型NR1-和nr2a亚基组成，在爪蟾卵母细胞或人胚胎肾（HEK） 293细胞中表达。在野生型通道中，Mg2+阻滞的电压依赖性（delta）与浓度相关，在0.01 mM时δ值接近0.58，在0.07 mM及更高浓度时δ值接近0.82。在胞外高Mg2+和高K+作为参比离子的生物离子条件下，Mg2+弱渗透通道。在中间电位范围内（类似于60 ~ -10 mV），这种弱磁导率对阻滞没有明显影响，但在负电位范围内（类似于60 mV），它减弱了阻滞的程度和对电压的依赖性。3.用甘氨酸、丝氨酸、谷氨酰胺或天冬氨酸取代nr1亚基中的n位天冬酰胺增强了中间电位上的阻滞程度，但使阻滞的电压依赖性保持不变，这表明阻滞的结构决定因素仍然存在。这些相同的取代或减弱或保持Mg2+的表观渗透率不变。在含有甘氨酸、丝氨酸或谷氨酰胺取代nr2a亚基n位天冬酰胺的通道中，Mg2+的阻滞在负电位下被还原。在中间电位上，除了谷氨酰胺取代外，阻滞没有强烈减弱，谷氨酰胺取代使阻滞的电压依赖性降低到0.7 mill Mg2+5中的0.57。nr2a亚基中N + 1位点天冬酰胺的等效取代在整个电压范围内强烈减弱了阻滞。在0.7 mM Mg2+中，阻滞的电压依赖性降低到0.50（甘氨酸）、0.53（丝氨酸）和0.46（谷氨酰胺）。nr2a亚基中含有N位或N + 1位天冬酰胺取代的通道显示Mg2+通透性增加，表明这些相邻的天冬酰胺形成了向内Mg2+通量的屏障。这一屏障的变化，至少在一定程度上促成了这些残基取代后阻滞破坏的潜在机制。邻近的nr2a亚基天冬酰胺位于或靠近狭窄的通道狭窄处。然而，孔隙大小并不能决定Mg2+阻断突变体通道的有效性。由此可见，在NMDA受体通道狭窄处，邻近的nr2a亚基天冬酰胺、N位点和N + 1位点，而不是nr1亚基的N位点天冬酰胺，形成了细胞外Mg2+的关键阻断位点。与主流观点相反，阻断位点对N + 1位点的贡献比N位点的天冬酰胺更强。阻滞可能涉及到Mg2+与这些残基的结合。
1. The voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set of different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild-type and mutant NR1- and NR2A-subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells.2. In wild-type channels, the voltage dependence (delta) of the block by Mg2+ was concentration dependent with values of delta of similar to 0.58 in 0.01 mM and similar to 0.82 in 0.07 mM and higher concentrations. Under biionic conditions with high extracellular Mg2+ and K+ as the reference ion, Mg2+ weakly permeated the channel. Over intermediate potentials (similar to-60 to -10 mV), this weak permeability had no apparent effect on the block but at potentials negative to similar to-60 mV, it attenuated the extent and voltage dependence of the block. 3.Substitutions of glycine, serine, glutamine or aspartate for the N-site asparagine in the NR1-subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg2+ permeability4.In channels containing substitutions of glycine, serine or glutamine for the N-site asparagine in the NR2A-subunit, the block by Mg2+ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to similar to 0.57 in 0.7 mill Mg2+5. Equivalent substitutions for the N + 1 site asparagine in the NR2A-subunit strongly attenuated the block over the entire voltage range. In 0.7 mM Mg2+, the voltage dependence of the block was reduced to 0.50 (glycine), 0.53 (serine) and 0.46 (glutamine).6. Channels containing substitutions of the N-site or N + 1 site asparagines in the NR2A-subunit showed an increased Mg2+ permeability suggesting that these adjacent asparagines form a barrier for inward Mg2+ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues.7. The adjacent NR2A-subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg2+ blocks mutant channels.8. It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A-subunit asparagines, the N-site and N + 1 site, but not the N-site asparagine of the NR1-subunit, form a critical blocking site for extracellular Mg2+. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N-site asparagine. The block may involve binding of Mg2+ to these residues.