No 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...

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca 2؉ and Mg 2؉ permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg 2؉ permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca 2؉ permeability along with concentration-dependent inhibition of Na ؉ -carrying currents by Ca 2؉ . Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca 2؉ -permeable TRPM channels, substantially increased Ca 2؉ permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca 2؉ permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.The melastatin subtype of transient receptor potential (TRPM) 6 ion channels is widely expressed in neuronal, cardiovascular, immune, and endothelial cells where they are engaged in diverse physiological and pathophysiological processes (1-7). TRPM2 channels are activated by adenosine diphosphoribose (ADPR) and also by oxidative stress and mediate immune function, insulin secretion, endothelial permeability, and cell death that are induced by oxidative stress (8 -14).All members of the transient receptor potential (TRP) channel superfamily, which includes TRPC, TRPV, TRPM, TRPP, TRPML, and TRPA subfamilies, have a basic architecture similar to voltage-gated potassium channels, with homo-or hetero-tetrameric arrangements around a central ion-conducting pore (1,7,15). Each subunit is considered to have intracellular N and C termini and six transmembrane segments (S1-S6) with a re-entrant pore loop connecting S5 and S6 (see Fig. 1A). Despite significant differences in the amino acid residue sequences of the pore loop among different TRP subfamilies, there are two stretches of amino acid residues that are thought to form the pore helix and the ion selectivity filter of these channels, respectively (15).TRPM channels, including TRPM2, show considerable permeability to Ca 2ϩ and other divalent cations, with the exception of TRPM4/5 channels that are selective for monovalent cations (8, 9, 13-18). The molecular basis for divalent cationic permeability of TRPM channels is not fully understood. Accumulating evidence supports an important role of the ion selectivity filter in Ca 2ϩ and Mg 2ϩ permeation of TRPV and TRPM channels (15, 1...

P2X(7) receptors are distinct from other ATP-gated P2X receptors in that they are potently inhibited by submicromolar concentrations of zinc and copper. The molecular basis for the strong functional inhibition by zinc and copper at this purinergic ionotropic receptor is controversial. We hypothesized that it involves a direct interaction of zinc and copper with residues in the ectodomain of the P2X(7) receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X(7) receptors, none of which is homologous to previously identified sites in other P2X receptors shown to be important for functional potentiation by zinc. We introduced alanine substitutions into each of these residues, expressed wild-type and mutated receptors in human embryonic kidney 293 cells, and recorded resulting ATP and BzATP-evoked membrane currents. Agonist concentration-response curves were similar for all 12 functional mutant receptors. Alanine substitution at His(62) or Asp(197) strongly attenuated both zinc and copper inhibition, and the double mutant [H62A/D197A] mutant receptor was virtually insensitive to inhibition by zinc or copper. Thus, we conclude that zinc and copper inhibition is due to a direct interaction of these divalent cations with ectodomain residues of the P2X(7) receptor, primarily involving combined interaction with His(62) and Asp(197) residues.

BACKGROUND AND PURPOSEThe 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 APPROACHThe 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 RESULTSThe 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 mM for ATP and 58 mM 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 IMPLICATIONSThe 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.