Roberts Jonathan A, Evans Richard J
Department of Cell Physiology & Pharmacology, University of Leicester, Leicester, UK.
J Neurochem. 2005 Mar;92(5):1256-64. doi: 10.1111/j.1471-4159.2004.02960.x.
Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed in Xenopus oocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-type and P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor.
脯氨酸残基在蛋白质的二级结构中可发挥重要作用。在P2X受体的细胞外ATP结合环中有四个完全保守的脯氨酸残基（以P2X1受体编号；P93、P166、P228和P272）以及三个保守性稍低的残基P196（七种亚型中的六种）、P174和P225（七种亚型中的五种）。我们对人P2X1受体中的单个保守脯氨酸残基进行了突变并确定了它们的特性。突变体在非洲爪蟾卵母细胞中表达，并使用双电极电压钳进行表征。与野生型相比，突变体P166A、P174A、P196A、P225A和P228A对ATP效力没有影响，而P93A的ATP效力降低了四倍。P272A、P272D和P272K受体突变体在细胞表面表达；然而，这些突变体无功能。相比之下，P272I、P272G和P272F产生了功能性通道，分别对ATP效力没有影响或增加了2.5倍或6.5倍。在P272F受体处，ATP类似物拮抗剂2',3'-O-(2,4,6-三硝基苯基)-ATP的表观亲和力增加了12.5倍。这些结果表明，单个脯氨酸残基对于正常的P2X受体功能不是必需的，并且P272周围的受体构象有助于ATP与受体结合。
