爪蟾
生物物理学
跨膜结构域
细胞生物学
细胞内
C端
环核苷酸结合域
生物
电压钳
生物化学
细胞外
化学
氨基酸
膜电位
肽序列
基因
作者
Anna Durner,Ellis Durner,Annette Nicke
出处
期刊:eLife
[eLife Sciences Publications, Ltd.]
日期:2023-01-04
卷期号:12
被引量:3
摘要
The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing “cap” domain, and a globular “ballast domain” containing GTP/GDP and dinuclear Zn 2+ -binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)–2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry. While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only 3 out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.
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