C-terminal Calmodulin-binding Motif Differentially Controls Human and Rat P2X7 Receptor Current Facilitation

Abstract

P2X 7 receptors (P2X 7 R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X 7 Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X 7 R resulted from a Ca 2؉ -calmodulin-dependent process and a distinct calcium-independent process. However, P2X 7 Rs show striking species differences; thus, this study compared the properties of ATPevoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X 7 R as well as rat/human, human/rat chimeric, and mutated P2X 7 Rs. Facilitation at the human P2X 7 R was 5-fold slower than at the rat P2X 7 R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X 7 R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X 7 R. Replacement of three critical residues of this binding domain from the rat into the human P2X 7 R (T541I, C552S, and G559V) reconstituted the Ca 2؉ -calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X 7 Rs. The absence of Ca 2؉ -dependent facilitation at the human P2X 7 R may represent a protective adaptation of the innate immune response in which P2X 7 R plays significant roles.

Intracellular Signaling Pathways Integrating the Pore Associated with P2X7R Receptor with Other Large Pores

“…We have shown in 2005 that intracellular Ca 2+ acts as a second messenger in the large conductance channel opening in peritoneal macrophages and 2BH4 cells (Faria et al, 2005). Similar data were found by other groups (Cankurtaran-Sayar et al, 2009;Roger et al, 2008Roger et al, , 2010Schachter et al, 2008), but others did not observe this effect (da Cruz et al, 2006;Iglesias et al, 2008;Schachter et al, 2008;Virginio et al, 1999). We continued to investigate the Ca 2+ participation in more detail, and we described a major Ca 2+ dependence in the P2X7R pore formation, but we also observed Ca 2+ independent events in the same cell types (Faria et al, 2010).…”

“…This result was also confirmed by other groups (Donnelly-Roberts et al, 2004;Faria et al, 2010;Lundy et al, 2004). However, Roger and coworkers (Roger et al, 2008(Roger et al, , 2010 reported that rat P2X7R induced large organic cation permeability ionic currents were dependent on critical residues of calmodulin binding domain when recorded in patch-clamp whole cell configuration. In this sense, the intracellular Ca 2+ concentration dependence in the P2X7R pore formation is still unclear.…”

P2X7 receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness

“…This property is a particularity of P2X 7 R and is called facilitation (Hibell et al, 2000;Roger et al, 2008Roger et al, , 2010a. This led to a seven times increase of the current density from the first to the eighth ATP application (Figure 2b).…”

“…Extracellular ATP acts as a signalling molecule by activating plasma membrane G protein-coupled P2Y receptors and/or ligand-gated ion channels P2X receptors (Burnstock, 2006). Among the members of the P2X receptors family, the latest cloned P2X 7 receptors (P2X 7 R) (Rassendren et al, 1997) are very unique by different features, some of them are: (1) their sensitivity to ATP (North, 2002), (2) their facilitation under successive or sustained applications of agonist (Hibell et al, 2000;Roger et al, 2008Roger et al, , 2010a and (3) the appearance of a large, non-selective membrane pore after sustained stimulations with ATP (Pelegrin and Surprenant, 2006). Recently, several tumours have been shown to express P2X 7 R at unusually high levels (Adinolfi et al, 2002;Zhang et al, 2004;Slater et al, 2004a, b;Wang et al, 2004a;Raffaghello et al, 2006;Deli et al, 2007;Solini et al, 2008), however, their functionality and involvement in the physiology of cancer cells remain unclear.…”

Ion Channels as Promising Therapeutic Targets for Melanoma

“…Extracellular ATP activates plasma membrane G protein-coupled P2Y receptors and/or ligand-gated cation-permeable channels (Na + / Ca 2+ / K + ) P2X receptors (Burnstock, 2006). Among the members of the P2X receptors family, the latest cloned P2X7 receptor (P2X7R) (Rassendren et al, 1997;Surprenant et al, 1996) is very unique in its functioning by many features, such as 1) its low sensitivity to ATP (North, 2002), 2) its increasing activity, called facilitation, under successive or sustained applications of agonist (Roger et al, 2010;Roger et al, 2008), and 3) the appearance of a large, non-selective membrane pore after sustained stimulations with ATP due to the P2X7-dependent activation of pannexin-1 (Pelegrin & Surprenant, 2006). From a physiological point of view P2X7R is expressed in cells from immune lineage and its activation by extracellular ATP at concentrations upper than 100 µM is considered as an alarming signal, and is a key step in the initiation of the inflammatory cascade through the NLP3 inflammasome (Di Virgilio, 2007;Pelegrin, 2008).…”