ATP increases murine neuroblastoma cell size through a PANX1- and macropinocytosis-dependent mechanism

Macropinocytosis is an endocytic process that allows cells to respond to changes in their environment by internalizing nutrients and cell surface proteins, as well as modulating cell size. Here, we identify that adenosine triphosphate (ATP) triggers macropinocytosis in murine Neuro2a neuroblastoma cells, driving an increase in cell size, and internalizing the ATP release channel pannexin 1 (PANX1) to macropinosomes. Amiloride treatment and mutation of an extracellular tryptophan (W74) in PANX1 abolished ATP-evoked cell area enlargement, suggesting that PANX1 may itself regulate this form of macropinocytosis. Transient expression of the GTP-hydrolysis resistant ADP-ribosylation factor 6 GTPase (ARF6 Q67L) led to increased cell size, PANX1 internalization and localization to endosomal compartments, consistent with macropinocytosis. Inhibiting macropinocytosis-associated GTPases, phosphoinositide-3 kinase (PI3K), and disrupting actin polymerization abolished ATP-induced PANX1 internalization, supporting a macropinocytic mechanism. Further, these inhibitors disrupted co-distribution of intracellular PANX1 with macropinosomal cargo. Several lipid-PANX1 interactions were identified with relevance to macropinocytic mechanisms. The role of PANX1 in ATP-mediated macropinocytosis could be particularly important for disease states implicating PANX1, such as cancer, where ATP can act as a purinergic regulator of cell growth/metastasis and as a supplementary energy source following internalization.