Kawajiri S, Dingledine R
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322.
Neuropharmacology. 1993 Nov;32(11):1203-11. doi: 10.1016/0028-3908(93)90014-t.
The voltage-dependent block of NMDA channels by Mg2+ is an important functional element of NMDA receptors, since relief of block by depolarization plays a key role in some forms of ischemic neurodegeneration and synaptic plasticity. To identify the relevant structural domains responsible for block by Mg2+ and TCP, we used site-directed mutagenesis to change individual amino acids of the rat NR1A subunit in a transmembrane region (599-DALTLSSAMWFSWGVLLNSGIGE-621, mutated residues underlined) previously shown to donate residues that influence ionic selectivity. Ten mutant NR1A subunits were co-expressed in Xenopus oocytes with either the epsilon 1 or NR2A subunits, and receptor properties were analyzed under two-electrode voltage clamp. The mutation N616R virtually abolished voltage-dependent Mg2+ block, reduced Zn2+ block 5-fold and greatly reduced Ba2+ permeability in confirmation of previous reports. This mutation also reduced the potency of TCP as a use-dependent blocker by 200-fold. The remaining low-affinity TCP block did not appear to be use-dependent, suggesting two blocking sites for TCP. None of the other mutations differed significantly from NR1A itself except S617N, which displayed a 6-fold reduction in Mg2+ block. A well-barrier model of permeation through the NMDA receptor channel is presented that quantitatively reproduces voltage-dependent Mg2+ block. This model demonstrates that only minimum changes energy profiles experienced by permeating ions, equivalent to the energy of a single hydrogen or ionic bond, are required to abolish Mg2+ block. These findings indicate that only small structural changes are needed to convert a Mg(2+)-insensitive ion channel to a channel with pronounced voltage-dependent Mg2+ block.
Mg2+对NMDA通道的电压依赖性阻断是NMDA受体的一个重要功能元件，因为去极化解除阻断在某些形式的缺血性神经退行性变和突触可塑性中起关键作用。为了确定负责Mg2+和TCP阻断的相关结构域，我们使用定点诱变来改变大鼠NR1A亚基在一个跨膜区域（599-DALTLSSAMWFSWGVLLNSGIGE-621，下划线为突变残基）中的单个氨基酸，该区域先前已显示会提供影响离子选择性的残基。将十个突变的NR1A亚基与ε1或NR2A亚基在非洲爪蟾卵母细胞中共表达，并在双电极电压钳下分析受体特性。N616R突变几乎完全消除了电压依赖性Mg2+阻断，使Zn2+阻断降低了5倍，并大大降低了Ba2+通透性，这与先前的报道一致。该突变还使TCP作为使用依赖性阻断剂的效力降低了200倍。其余低亲和力的TCP阻断似乎不是使用依赖性的，这表明TCP有两个阻断位点。除了S617N使Mg2+阻断降低了6倍外，其他突变与NR1A本身均无显著差异。本文提出了一个通过NMDA受体通道的渗透势垒模型，该模型定量再现了电压依赖性Mg2+阻断。该模型表明，只需使渗透离子所经历的能量分布发生最小变化，相当于单个氢键或离子键的能量，就能消除Mg2+阻断。这些发现表明，只需进行微小的结构改变，就能将一个对Mg(2+)不敏感的离子通道转变为一个具有明显电压依赖性Mg2+阻断的通道。
