Polar Residues in the Second Transmembrane Domain of the Rat P2X2 Receptor That Affect Spontaneous Gating, Unitary Conductance, and Rectification
Polar Residues in the Second Transmembrane Domain of the Rat P2X2 Receptor That Affect Spontaneous Gating, Unitary Conductance, and Rectification
复制标题
DOI：
10.1523/jneurosci.4403-09.2009
复制
发表时间：
2009-11-11
影响因子：
5.3
通讯作者：
North, R. Alan
中科院分区：
文献类型：
--
作者：
Cao, Lishuang;Broomhead, Helen E.;North, R. Alan
细胞外ATP（P2X受体）激活的膜离子通道广泛分布于神经系统中。它们的分子结构从根本上不同于烟碱或谷氨酸受体家族。我们测量了大鼠 P2X2 受体的单通道电流、自发门控和整流，其中通过诱变系统地探测了第二跨膜结构域 (TM2) 的极性和带电残基。结果表明Asn(333)和Asp(349)分别位于外前庭和内前庭。 Asn(333)、Thr(336) 和 Ser(340) 的取代特别可能引起自发激活通道。在 Thr(336)、Thr(339) 和 Ser(340) 处，引入正电荷（Arg、Lys、His 或 Cys，然后用 2-氨乙基甲硫磺酸盐处理）大大增强了外向电流，表明这三个残基的侧链暴露在开放通道的渗透途径中。这些功能发现是在最近报道的处于闭合状态的斑马鱼 P2X4.1 受体的 3.1 埃晶体结构的背景下解释的。它们暗示门是由残基 Asn333 至 Thr339 形成的，并且通道打开涉及逆时针旋转和 TM2 螺旋的分离。
Membrane ion channels activated by extracellular ATP (P2X receptors) are widely distributed in the nervous system. Their molecular architecture is fundamentally distinct from that of the nicotinic or glutamate receptor families. We have measured single-channel currents, spontaneous gating, and rectification of rat P2X2 receptor in which polar and charged residues of the second transmembrane domain (TM2) were systematically probed by mutagenesis. The results suggest that Asn(333) and Asp(349) lie respectively in external and internal vestibules. Substitutions at Asn(333), Thr(336), and Ser(340) were particularly likely to cause spontaneously active channels. At Thr(336), Thr(339), and Ser(340), the introduction of positive charge (Arg, Lys, or His, or Cys followed by treatment with 2-aminoethyl methanethiosulphonate) greatly enhanced outward currents, suggesting that side-chains of these three residues are exposed in the permeation pathway of the open channel. These functional findings are interpreted in the context of the recently reported 3.1 angstrom crystal structure of the zebrafish P2X4.1 receptor in the closed state. They imply that the gate is formed by residues Asn333 to Thr339 and that channel opening involves a counter-clockwise rotation and separation of the TM2 helices.