Rassendren F, Buell G, Newbolt A, North R A, Surprenant A
Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Switzerland.
EMBO J. 1997 Jun 16;16(12):3446-54. doi: 10.1093/emboj/16.12.3446.
P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.
P2X受体是由细胞外ATP开启的离子通道。目前已知的七个亚基由不同基因编码。据认为，每个亚基有两个跨膜结构域、一个大的细胞外环以及细胞内的N端和C端，这种拓扑结构与其他配体门控通道（如烟碱型乙酰胆碱或谷氨酸受体）的拓扑结构根本不同。我们使用半胱氨酸替代可及性方法来确定构成P2X2受体离子孔的分子部分。将第二个疏水结构域（316 - 354）之前及整个区域的氨基酸逐个突变为半胱氨酸，并将这些DNA在HEK293细胞中表达。对于38个残基中的3个（I328C、N333C、T336C），ATP诱发的电流可被细胞外施加的任何一种电荷的甲硫基磺酸盐（乙基三甲基铵、乙磺酸盐）抑制，这表明它们位于孔的外前庭。对于另外两个替代位点（L338C、D349C），只有较小的乙胺衍生物能抑制电流。无论通道是否由ATP开启，L338C都可被半胱氨酸修饰，但D349C仅在同时施加ATP时受到抑制。结果表明，P2X受体孔的一部分由第二个疏水结构域形成，且L338和D349位于通道“门”的两侧。
