Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors
Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors
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DOI：
10.1007/s00249-008-0325-9
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发表时间：
2009-03-01
影响因子：
2
通讯作者：
Nicke, Annette
中科院分区：
文献类型：
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作者：
Marquez-Klaka, Benjamin;Rettinger, Jurgen;Nicke, Annette
P2 X受体是ATP门控阳离子通道，由7个克隆的亚基组装成同源三聚体或异源三聚体。每个亚基含有两个跨膜结构域，由一个大的细胞外环连接。我们先前已经证明，用半胱氨酸残基取代两个保守残基K68和F291，导致相邻P2 X（1）亚基之间的二硫键交联。由于这些残基的突变导致ATP效力降低，并且在ATP存在下阻止半胱氨酸交联，因此我们提出了亚基间ATP结合位点。为了研究这些残基的邻近性是否在其他P2 X亚型中保留，我们测试了相应的P2 X（2）（K69 C，F289 C）、P2 X（3）（K63 C，F280 C）和P2 X（4）（K67 C，F294 C）突变体在非洲爪蟾卵母细胞中成对表达后的自发胱氨酸形成。纯化受体的非还原SDS-PAGE分析显示P2 X（2）K69 C和P2 X（2）F289 C突变体之间特异性二聚体形成。同样地，P2 X（1）K68 C和P2 X（2）F289 C的共表达，而不是P2 X（1）F291 C和P2 X（2）K69 C突变体的共表达导致相应亚基之间的二聚体形成。交联的P2 X（1/2）异聚体显示出强烈降低或缺失的功能，其在用DTT处理后选择性地恢复。P2 X（3）或P2 X（4）突变体之间的交联效率较低，但可通过短半胱氨酸反应性交联剂MTS-2-MTS增强。这些结果表明，与P2 X（1）中的位置K68和F291类似的残基的空间邻近性和/或取向在P2 X（2）受体中以及在异聚体P2 X（1/2）受体中的两个可能的界面之一处被保留，但对于P2 X（3）和P2 X（4）受体功能似乎是多余的。
P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulfide cross-linking between neighbouring P2X(1) subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X(2) (K69C, F289C), P2X(3) (K63C, F280C), and P2X(4) (K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the purified receptors revealed a specific dimer formation between P2X(2)K69C and P2X(2)F289C mutants. Likewise, co-expression of P2X(1)K68C and P2X(2)F289C, but not P2X(1)F291C and P2X(2)K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X(1/2) heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Cross-linking was less efficient between P2X(3) or P2X(4) mutants but could be enhanced by the short cysteine-reactive cross-linker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X(1) are preserved in P2X(2) receptors and at one of two possible interfaces in heteromeric P2X(1/2) receptors but appears to be redundant for P2X(3) and P2X(4) receptor function.