The large intracellular C-terminus of the pro-inflammatory P2×7 ion channel receptor (P2×7R) is associated with diverse P2×7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2×7R 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 P2×7Rs and performed voltage clamp fluorometry (VCF). While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only three 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.