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Key points: Re‐sensitization of P2X4 receptors depends on a protonation/de‐protonation cycleProtonation and de‐protonation of the receptors is achieved by internalization and recycling of P2X4 receptors via acidic compartmentsProtonation and de‐protonation occurs at critical histidine residues within the extracellular loop of P2X4 receptorsRe‐sensitization is blocked in the presence of the receptor agonist ATP P2X4 receptors are members of the P2X receptor family of cation‐permeable, ligand‐gated ion channels that open in response to the binding of extracellular ATP. P2X4 receptors are implicated in a variety of biological processes, including cardiac function, cell death, pain sensation and immune responses. These physiological functions depend on receptor activation on the cell surface. Receptor activation is followed by receptor desensitization and deactivation upon removal of ATP. Subsequent re‐sensitization is required to return the receptor into its resting state. Desensitization and re‐sensitization are therefore crucial determinants of P2X receptor signal transduction and responsiveness to ATP. However, the molecular mechanisms controlling desensitization and re‐sensitization are not fully understood. In the present study, we provide evidence that internalization and recycling via acidic compartments is essential for P2X4 receptor re‐sensitization. Re‐sensitization depends on a protonation/de‐protonation cycle of critical histidine residues within the extracellular loop of P2X4 receptors that is mediated by receptor internalization and recycling. Interestingly, re‐sensitization under acidic conditions is completely revoked by receptor agonist ATP. Our data support the physiological importance of the unique subcellular distribution of P2X4 receptors that is predominantly found within acidic compartments. Based on these findings, we suggest that recycling of P2X4 receptors regulates the cellular responsiveness in the sustained presence of ATP. Key points: Re‐sensitization of P2X4 receptors depends on a protonation/de‐protonation cycleProtonation and de‐protonation of the receptors is achieved by internalization and recycling of P2X4 receptors via acidic compartmentsProtonation and de‐protonation occurs at critical histidine residues within the extracellular loop of P2X4 receptorsRe‐sensitization is blocked in the presence of the receptor agonist ATP [ABSTRACT FROM AUTHOR]

Rho GTPases play prominent roles in the regulation of cytoskeletal reorganization. Many aspects have been elaborated concerning the individual functions of Rho GTPases in distinct signaling pathways leading to cytoskeletal rearrangements. However, major questions have yet to be answered regarding the integration and the signaling hierarchy of different Rho GTPases in regulating the cytoskeleton in fundamental physiological events like neuronal process differentiation. Here, we investigate the roles of the small GTPases Rac1, Cdc42, and RhoG in defining dendritic tree complexity stimulated by the transmembrane epidermal growth factor family member CALEB/ NGC. Combining gain-of-function and loss-of-function analysis in primary hippocampal neurons, we find that Rac1 is essential for CALEB/ NGC-mediated dendritic branching. Cdc42 reduces the complexity of dendritic trees. Interestingly, we identify the palmitoylated isoform of Cdc42 to adversely affect dendritic outgrowth and dendritic branching, whereas the prenylated Cdc42 isoform does not. In contrast to Rac1, CALEB/ NGC and Cdc42 are not directly interconnected in regulating dendritic tree complexity. Unlike Rac1, the Rac1-related GTPase RhoG reduces the complexity of dendritic trees by acting upstream of CALEB/ NGC. Mechanistically, CALEB/ NGC activates Rac1, and RhoG reduces the amount of CALEB/ NGC that is located at the right site for Rac1 activation at the cell membrane. Thus, Rac1, Cdc42, and RhoG perform very specific and non-redundant functions at different levels of hierarchy in regulating dendritic tree complexity induced by CALEB/ NGC. [ABSTRACT FROM AUTHOR]

Two fundamental mechanisms within alveoli are essential for lung function: regulated fluid transport and secretion of surfactant. Surfactant is secreted via exocytosis of lamellar bodies (LBs) in alveolar type II (ATII) cells. We recently reported that LB exocytosis results in fusion-activated cation entry (FACE) via P2X4 receptors on LBs. We propose that FACE, in addition to facilitating surfactant secretion, modulates alveolar fluid transport. Correlative fluorescence and atomic force microscopy revealed that FACE-dependent water influx correlated with individual fusion events in rat primary ATII cells. Moreover, ATII cell monolayers grown at air-liquid interface exhibited increases in short-circuit current (Isc) on stimulation with ATP or UTP. Both are potent agonists for LB exocytosis, but only ATP activates FACE. ATP, not UTP, elicited additional fusion-dependent increases in Isc. Overexpressing dominant-negative P2X4 abrogated this effect by ~50%, whereas potentiating P2X4 lead to ~80% increase in Isc. Finally, we monitored changes in alveolar surface liquid (ASL) on ATII monolayers by confocal microscopy. Only stimulation with ATP, not UTP, led to a significant, fusion-dependent, 20% decrease in ASL, indicating apical-to-basolateral fluid transport across ATII monolayers. Our data support the first direct link between LB exocytosis, regulation of surfactant secretion, and transalveolar fluid resorption via FACE. [ABSTRACT FROM AUTHOR]