Contribution of conserved glycine residues to ATP action at human P2X1 receptors:: mutagenesis indicates that the glycine at position 250 is important for channel function
Contribution of conserved glycine residues to ATP action at human P2X1 receptors:: mutagenesis indicates that the glycine at position 250 is important for channel function
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DOI：
10.1111/j.1471-4159.2005.03494.x
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发表时间：
2005-12-01
影响因子：
4.7
通讯作者：
Evans, RJ
中科院分区：
文献类型：
--
作者：
Digby, HR;Roberts, JA;Evans, RJ
甘氨酸残基可以引入蛋白质的灵活性，引起二级结构的转折和断裂，是一些核苷酸结合基序的关键组成部分。在P2X受体胞外ATP结合域中，11个甘氨酸残基是完全保守的，另外5个残基在7个家族成员中至少有5个是保守的。我们突变了单个保守的甘氨酸残基，并测定了它们对非洲爪哇卵母细胞表达的P2X(1)受体的ATP敏感性和时程的影响。在大多数情况下，丙氨酸替代对ATP敏感性和反应时间进程没有或不到3倍的影响。G71A使ATP活性降低6倍，10 mM的ATP不能引起G96A、G250A和G301A突变型受体的功能反应。然而，在第96位和第301位，脯氨酸或半胱氨酸可以取代甘氨酸，得到基本上正常的受体。当甘氨酸250被丝氨酸取代时，对ATP的功能反应对ATP敏感性没有影响，但峰值降低；相反，当甘氨酸250被丙氨酸、半胱氨酸、天冬氨酸、苯丙氨酸、异亮氨酸、赖氨酸、脯氨酸或天冬氨酸取代时，没有记录到功能反应。这些结果表明，甘氨酸250在决定P2X受体的功能中起着重要作用。
Glycine residues can introduce flexibility in proteins, give rise to turns and breaks in secondary structure and are key components of some nucleotide binding motifs. In the P2X receptor extracellular ATP binding domain, 11 glycine residues are completely conserved and an additional five are conserved in at least five of the seven family members. We have mutated individual conserved glycine residues and determined their effect on the ATP sensitivity and time-course of P2X(1) receptors expressed in Xenopus oocytes. In the majority of cases, replacement by alanine had no or a less than 3-fold effect on ATP sensitivity and time-course of responses. G71A resulted in a 6-fold decrease in ATP potency and ATP (10 mM) failed to evoke functional responses from G96A, G250A and G301A mutant receptors. However, proline or cysteine could substitute for glycine at positions 96 and 301, giving receptors that were essentially normal. At glycine 250 substitution by serine gave functional responses to ATP with no effect on ATP sensitivity but a reduction in peak amplitude; in contrast, functional responses were not recorded when glycine 250 was replaced by the amino acids alanine, cysteine, aspartate, phenylalanine, isoleucine, lysine, proline or asparagine. These results suggest that glycine 250 plays an important role in determining the function of P2X receptors.