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 single-channel function, respectively. Such interpretations are consistent with the locations of the residues in the structural model of the hP2X(7) receptor.
Highlights
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 amplitude of currents mediated by the hP2X7 receptor was lower than that mediated by the rP2X7 receptor
By combining site-directed mutagenesis with whole-cell current patch-clamp recording and protein expression assays, we have shown that the residues at positions 155 and 348 are critical in determining ATP-induced currents mediated by P2X7 receptors, as well as the difference between the human and rat receptors
Summary
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 functional expression level of P2X7 receptor is crucial in its role in the disease pathogenesis. Our recent study characterizing the mutations resulting from non-synonymous SNPs in the human P2RX7 gene has shown that H155Y and A348T mutations increase ATP-induced currents [53]. These two gain-of-function mutations substitute the residues in the hP2X7 receptor with the corre-. We show evidence for a critical role of residues at positions 155 and 348 in determining the ATP-induced currents mediated by hP2X7 and rP2X7 receptors and the distinct mechanisms that underlie their contribution
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call