Residues 155 and 348 contribute to the determination of P2X7 receptor function via distinct mechanisms revealed by single-nucleotide polymorphisms.

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 single-channel function, respectively. Such interpretations are consistent with the locations of the residues in the structural model of the hP2X(7) receptor.

Pharmacological properties of the rhesus macaque monkey P2X7 receptor.

BACKGROUND AND PURPOSE: The human P2X7 (hP2X7) receptor exhibits striking pharmacological differences from its rodent counterparts, particularly in terms of its antagonist profile. Here, we characterized the functional and pharmacological properties of the rhesus macaque monkey P2X7 (rmP2X7) receptor in comparison with the hP2X7 receptor. EXPERIMENTAL APPROACH: The rmP2X7 and hP2X7 receptors were heterologously expressed in HEK293 cells. The receptor surface and total expression levels were examined by biotin-labelling and Western blotting. The functional and pharmacological properties were characterized using patch-clamp recording and single-cell imaging. KEY RESULTS: The rmP2X7 receptor showed strong cell surface expression. Both ATP and 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) were full agonists in activating the rmP2X7 receptor; the EC50 values were 802 µM for ATP and 58 µM for BzATP, respectively, in extracellular low divalent cation solution. Prolonged activation of the rmP2X7 receptors induced detectable but low level YO-PRO-1 uptake. KN-62, AZ11645373 and A-438079, three hP2X7 selective antagonists, all potently inhibited the rmP2X7 receptor-mediated currents; the IC50 values were 86, 23 and 297 nM respectively. CONCLUSION AND IMPLICATIONS: The rmP2X7 receptor exhibits similar pharmacological properties to the hP2X7 receptor. The rhesus macaque monkey thus may represent a valuable model species in elucidating the mechanisms and pharmacological interventions of hP2X7 receptor-related diseases.