P2X 7 receptors are important in mediating the physiological functions of extracellular ATP, and altered receptor expression and function have a causative role in the disease pathogenesis. Here, we investigated the mechanisms determining the P2X 7 receptor function by following two human single-nucleotide polymorphism (SNP) mutations that replace His-155 and Ala-348 in the human (h) P2X 7 receptor with the corresponding residues, Tyr-155 and Thr-348, in the rat (r) P2X 7 receptor. H155Y and A348T mutations in the hP2X 7 receptor increased ATP-induced currents, whereas the reciprocal mutations, Y155H and T348A, in the rP2X 7 receptor caused the opposite effects. Such a functional switch is a compelling indication that these residues are critical for P2X 7 receptor function. Additional mutations of His-155 and Ala-348 in the hP2X 7 receptor to residues with diverse side chains revealed a different dependence on the side chain properties, supporting the specificity of these two residues. Substitutions of the residues surrounding His-155 and Ala-348 in the hP2X 7 receptor with the equivalent ones in the rP2X 7 receptor also affected ATP-induced currents but were not fully reminiscent of the H155Y and A348T effects. Immunofluorescence imaging and biotin labeling assays showed that H155Y in the hP2X 7 receptor increased and Y155H in the rP2X 7 receptor decreased cell-surface expression. Such contrasting effects were not obvious with the reciprocal mutations of residue 348. Taken together, our results suggest that residues at positions 155 and 348 contribute to P2X 7 receptor function via determining the surface expression and the singlechannel function, respectively. Such interpretations are consistent with the locations of the residues in the structural model of the hP2X 7 receptor. P2X 7 receptors belong to the ionotropic purinergic P2X receptor family (1-5). The receptor expression is well documented in immune cells, glial cells in the brain, satellite cells in the peripheral nervous system, bone, and epithelial cells, where the receptor serves as the primary mediator for numerous physiological functions of extracellular ATP, including immune responses, inflammation, cell proliferation, neuron-glial cell interactions, nociception, bone remodeling, and saliva secretion (6 -17). The receptor is thought to be a homotrimer (18) with three subunits intertwining together along a vertical ionconducting pathway. Each subunit contains two transmembrane domains (TM1 and TM2) joined by a large ectodomain, and both N and C termini reside intracellularly. Functional characterizations of mammalian P2X 7 receptors have identified several distinguishing features, including activation by submillimolar concentrations of ATP and a higher sensitivity to benzoylbenzoyl-ATP (BzATP), 4 a synthetic ATP analog, than to ATP (19 -21). In addition to operating as ATP-gated Ca 2ϩ -permeable cation channels that open within milliseconds upon agonist binding, P2X 7 receptors induce formation of a large pore, activation of the inflammasome, and n...

a b s t r a c tWe investigated membrane targeting of the P2X 7 receptor (P2X 7 R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein l1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X 7 R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process. Structured summary:MINT-8055849:Beta-catenin (uniprotkb:B6V8E6) and P2X7R (uniprotkb:Q64663) colocalize (MI:0403) by fluorescence microscopy (MI:0416)