Institute of Physiology, Czech Academy of Sciences, 14220 Prague, Czech Republic.
1st Faculty of Medicine, Charles University, 12108 Prague, Czech Republic.
Int J Mol Sci. 2020 Nov 10;21(22):8446. doi: 10.3390/ijms21228446.
Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.
P2X7 受体的激活会导致一个大孔的打开，该孔在免疫反应、细胞凋亡和许多其他生理和病理过程中发挥作用。在这里，我们研究了在连接激动剂结合域和跨膜域的大鼠 P2X7 受体的细胞外前庭中的保守和独特残基的作用。我们发现，所有在 P2X 受体亚型中保守的残基对丙氨酸诱变的反应是抑制（Y51、Q52 和 G323）或显著降低（K49、G326、K327 和 F328）2'，3'-O-（苯甲酰-4-苯甲酰）-ATP（BzATP）诱导的电流和溴化乙锭的通透性，而在 P2X4 受体中也存在的非保守残基（F322）对 BzATP 的敏感性提高了 10 倍，失活动力学慢得多，并且形成大染料渗透性孔的倾向更高。我们检查了保守突变体的膜表达，发现 Y51、Q52、G323 和 F328 对受体向质膜的运输起作用，而 K49 控制受体对激动剂的反应性。最后，我们研究了这些残基的物理化学性质的重要性，并观察到 K49R、F322Y、F322W 和 F322L 突变体显著逆转了受体功能，表明带正电荷和大疏水性残基分别在位置 49 和 322 很重要。这些结果表明，跨膜域 1（K49-Y51-Q52）和跨膜域 2（G326-K327-F328）上方的保守残基簇对于受体结构、膜表达和通道门控很重要，而细胞外前庭顶部的非保守残基（F322）参与疏水性亚基间相互作用，稳定 P2X7 受体通道的关闭状态。
