Residues 155 and 348 Contribute to the Determination of P2X7 Receptor Function via Distinct Mechanisms Revealed by Single-nucleotide Polymorphisms

Abstract

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...

Gain and Loss of Function of P2X7 Receptors: Mechanisms, Pharmacology and Relevance to Diabetic Neuropathic Pain

“…While conflicting results are present in the literature for this specific SNP, our data are consistent with [23,27] showing that the 348Thr variant also generates a gain of function phenotype.…”

“…However, significant differences in maximum responses were identified for both modes of activation (178 ± 6% and 126 ± 12% for pore and channel function, respectively, Table  1), confirming previous findings of a GOF phenotype driven by 155Tyr [22,23,25-27]. …”

The P2X7 Receptor Channel: Recent Developments and the Use of P2X7 Antagonists in Models of Disease

“…The underlying mechanisms behind these gain-of-function effects have not been established. There is some evidence to suggest that the amino acid variation at 155 increases cell surface expression of P2X7 (Bradley et al, 2011a); however, this was not observed in the initial report of this SNP (Cabrini et al, 2005). One of the most interesting SNPs in human P2RX7 is Q460R in the C terminus.…”

Purinergic P2X Receptors: Structural and Functional Features Depicted by X-Ray and Molecular Modelling Studies

“…Roger et al mapped single nucleotide polymorphisms identified in affective mode disorders on to the model, and were able to suggest mechanisms by which the mutations caused impairment in channel function [112]. This model was also used in a further study analysing two gain-of-function SNPs in human P2X7 (H155Y and A348T), comparing them with their reciprocal mutations in rat P2X7 [113]. Finally, the same model was used in a paper comparing the function of the highly homologous human and rhesus macaque P2X7…”