A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics
A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics
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DOI：
10.1111/j.1469-7793.1999.0315v.x
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发表时间：
1999-04-15
影响因子：
5.5
通讯作者：
Auerbach, A
中科院分区：
文献类型：
--
作者：
Salamone, FN;Zhou, M;Auerbach, A
1.在我们的小鼠肌肉α亚基乙酰胆碱受体通道（AChR）cDNA克隆的常规测序，我们发现了与GenBank数据库条目（登录X 03986）的差异。在核苷酸1305-7（残基433，在M4结构域中），数据库列出了编码缬氨酸的GTC，而我们假定的“野生型”cDNA具有编码丙氨酸的核苷酸GCC。未发现其它序列差异.基因组DNA的PCR扩增证实BALB/C小鼠α亚基基因在位置1306处具有T核苷酸，并且因此，该蛋白质在M4区段中的位置433处具有V。为了确定这种差异的功能后果，野生型（V433）或突变体（A433）α亚基在HEK细胞中与小鼠β、β和δ亚基共表达。单通道电流记录在细胞附着的补丁，和速率和平衡常数估计从开放和关闭的持续时间从一系列的乙酰胆碱浓度。V433和A433变体的活化速率常数和平衡常数之间没有显著差异.α V433 AChR的动力学模型表明，两个递质结合位点对乙酰胆碱具有相似的解离平衡常数（类似于142 mM细胞外KCl中的160 μ M）。双配体的乙酰胆碱受体占据一个封闭的状态，具有类似于1毫秒的寿命。进入和离开这个状态的速率常数不随乙酰胆碱浓度而变化。一个突变型乙酰胆碱受体，导致慢通道先天性肌无力综合征，α G153 S，动力学进行了重新检查。该突变体的性质与α 433位的V或A相似。
1. During routine sequencing of our mouse muscle alpha subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in the M4 domain) the database lists GTC which encodes a valine, while our putative 'wild-type' cDNA had the nucleotides GCC, which encodes an alanine. No other sequence differences were found.2. PCR amplification of genomic DNA confirmed that the BALB/C mouse alpha subunit gene has a T nucleotide at position 1306, and, therefore, that the protein has a V at position 433 in the M4 segment.3. In order to determine the functional consequences of this difference, either wild-type (V433) or mutant (A433) alpha subunits were co-expressed in HEK cells with mouse beta, epsilon and delta subunits. Single-channel currents were recorded in cell-attached patches, and rate and equilibrium constants were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activation rate constants or equilibrium constants of the V433 and A433 variants.4. Kinetic modelling of alpha V433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine (similar to 160 mu M, in 142 mM extracellular KCl).5. Diliganded AChRs occupy a closed state that has a lifetime of similar to 1 ms. The rate constants for entering and leaving this state do not vary with the ACh concentration.6. The kinetics of a mutant AChR that causes a slow channel congenital myaesthenic syndrome, alpha G153S, was re-examined. The properties of this mutant were similar with a V or an A at position alpha 433.