Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2012 Jun 22;287(26):22099-111. doi: 10.1074/jbc.M112.369157. Epub 2012 May 3.
Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2-100 μM, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μM. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nM) to very low (>100 μM) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.
人 P2X2 受体（hP2X2）在 2-100 μM 的范围内强烈被锌抑制，而大鼠 P2X2 受体（rP2X2）在同一范围内强烈被锌增强，然后在超过 100 μM 的锌中被抑制。然而，在这两个物种中，锌调节的生物学作用尚不清楚。为了确定控制 hP2X2 中锌抑制的候选区域，根据斑马鱼 P2X4.1 的晶体结构构建了同源模型。在该模型中，一个亚基的 His-204 和 His-209 与相邻亚基的 His-330 接近。交联研究证实，这些残基彼此之间的距离在 8 Å 以内。同时将这三个组氨酸突变为丙氨酸，使 hP2X2 的锌抑制作用降低近 100 倍。在 rP2X2 中，这些组氨酸中的一个被赖氨酸取代，并且在消除锌增强的背景下，将 Lys-197 突变为组氨酸将 rP2X2 从低效力转变为高效力抑制。我们探讨了锌结合位点是否位于沿着受体中心轴运行的前庭中。从上前庭中消除所有带负电荷的残基对锌抑制没有影响。相比之下，hP2X2 中前庭中部的几个残基的突变导致锌抑制效力的显著变化。特别是，P206C 的锌效力可以通过 MTSET 的结合和解离从非常高（约 10 nM）可逆地转变为非常低（>100 μM）。这些结果表明，亚基界面处的组氨酸簇控制锌进入其结合位点的通道。
