Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors.
Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors.
复制标题
半胱氨酸取代突变体给出结构洞察力，并鉴定P2X受体处的ATP结合和激活位点。
DOI：
10.1523/jneurosci.2310-06.2007
复制
发表时间：
2007-04-11
影响因子：
5.3
通讯作者：
Evans, Richard J
中科院分区：
文献类型：
--
作者：
Roberts, Jonathan A;Evans, Richard J
细胞外ATP的P2 X受体是一个独特的配体门控阳离子通道家族，参与从突触传递到肌肉收缩的生理过程。常见的ATP结合基序不存在于P2 X受体中，激动剂结合位点的程度尚不清楚。我们使用半胱氨酸扫描诱变，放射性标记的2-叠氮ATP结合，和甲硫基磺酸盐（MTS）化合物，以确定参与ATP结合和门控的人P2 X1受体的氨基酸残基。MTSEA-生物素化的模式也用于确定取代的半胱氨酸残基的可及性以及这是否在添加ATP时发生变化。对第二个跨膜段之前的胞外环中最后44个氨基酸残基（S286-I329）的半胱氨酸取代突变体的分析表明，N290、F291、R292和K309突变体具有降低的ATP效力和2-叠氮基ATP结合。MTS试剂在这些残基处产生ATP效价的进一步变化，表明它们直接参与ATP结合;该效应取决于MTS试剂在K309 C处的电荷，对此的一种解释是K309直接与ATP的带负电荷的磷酸盐相互作用。其余的半胱氨酸取代对ATP效力几乎没有影响。然而，在突变体D316 C、G321 C、A323 C和I328 C处，MTS试剂不改变ATP效力，但改变激动剂诱发的反应，表明该区域可能有助于通道的门控。
P2X receptors for extracellular ATP are a distinct family of ligand gated cation channels involved in physiological processes ranging from synaptic transmission to muscle contraction. Common ATP binding motifs are absent from P2X receptors and the extent of the agonist binding site is unclear. We used cysteine scanning mutagenesis, radiolabelled 2-azido ATP binding, and methanethiosulfonate (MTS) compounds, to identify amino acid residues involved in ATP binding and gating of the human P2X1 receptor. The pattern of MTSEA-biotinylation was also used to determine the accessibility of substituted cysteine residues and whether this changed on addition of ATP. Analysis of cysteine substituted mutants of the last 44 amino acid residues (S286-I329) in the extracellular loop before the second transmembrane segment showed that N290, F291, R292 and K309 mutants had reduced ATP potency and 2-azido ATP binding. MTS reagents produced further shifts in ATP potency at these residues suggesting that they are directly involved in ATP binding; the effects were dependent on the charge of the MTS reagent at K309C, one explanation for this is that K309 interacts directly with the negatively charged phosphate of ATP. The remainder of the cysteine substitutions had little or no effect on ATP potency. However at the mutants D316C, G321C, A323C, and I328C MTS reagents did not change ATP potency but modified agonist evoked responses suggesting that this region may contribute to the gating of the channel.