Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors

ATP-gated purinergic P2X4 receptors (P2X4Rs) are the most alcohol-sensitive P2XR subtype. We recently reported that ivermectin (IVM), an antiparasitic used in animals and humans, antagonized ethanol inhibition of P2X4Rs. Furthermore, IVM reduced ethanol intake in mice. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed an action pocket for both ethanol and IVM formed by Asp331, Met336 in TM2 and Trp46, and Trp50 in TM1 segments. The role of Asp331 and Met336 was experimentally confirmed. The present study tested the hypothesis that Trp46 plays a role in ethanol and IVM modulation of P2X4Rs. Trp46 was mutated to residues with different physicochemical properties and the resultant mutants tested for ethanol and IVM responses using Xenopus oocyte expression system and two-electrode voltage clamp. Nonaromatic substitutions at position 46 reduced ethanol inhibition at higher concentrations and switched IVM potentiation to inhibition. Simultaneous substitution of alanine at positions Trp46 and Met336 also resulted in similar changes in ethanol and IVM responses. Furthermore, a new molecular model based on the open pore conformation of zebrafish P2X4R suggested a role for Tyr42 that was further supported experimentally. Our previous and current findings, combined with our preliminary evidence of increased ethanol consumption in P2X4R knockout mice, suggest that the ethanol and IVM action pocket in P2X4Rs formed by positions 42, 46, 331, and 336 presents a potential target for medication development for alcohol use disorders.     

Neurosteroids differentially modulate P2X4 ATP-gated channels through non-genomic interactions

As neuroactive steroids modulate several ionotropic receptors, we assessed whether the ATP-gated currents elicited by P2X₄ receptors are modulated by these compounds. We transfected HEK293 cells or injected Xenopus laevis oocytes with the cDNA coding for rat P2X₄ receptor. Application of 0.1–10 μM alfaxolone potentiated within 60-s the 1 μM ATP-evoked currents with a maximal potentiation of 1.8 and 2.6-fold in HEK293 or oocytes cells respectively. Allopregnalolone or 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) also potentiated the ATP-gated currents but with a maximal effect only averaging 1.25 and 1.35-fold respectively. In contrast, 0.3–10 μM pregnanolone, but not its sulfated derivative, inhibited the ATP-gated currents; the maximal inhibition reached 40% in both cell types. THDOC, but not other neurosteroids increased significantly the τ_off of the ATP-evoked currents, revealing another mode of neurosteroid modulation. Sexual steroids such as 17β-estradiol or progesterone were inactive revealing explicit structural requirements. Alfaxolone or THDOC at concentrations 30- to 100-fold larger than required to modulate the receptor, gated the P2X₄ receptor eliciting ATP-like currents that were reduced with suramin or brilliant blue G, but potentiated the P2X₄ receptor more than 10-fold by 10 μM zinc. In conclusion, neurosteroids rapidly modulate via non-genomic mechanisms and with nanomolar potencies, the P2X4 receptor interacting likely at distinct modulator sites.     

The effect of anions on the human P2X7 receptor

P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4-liganded P2X7R through an extracellular anion binding site.     

P2X4 purinoceptor signaling in chronic pain

ATP, acting via P2 purinergic receptors, is a known mediator of inflammatory and neuropathic pain. There is increasing evidence that the ATP-gated P2X4 receptor (P2X4R) subtype is a locus through which activity of spinal microglia and peripheral macrophages instigate pain hypersensitivity caused by inflammation or by injury to a peripheral nerve. The present article highlights the recent advances in our understanding of microglia-neuron interactions in neuropathic pain by focusing on the signaling and regulation of the P2X4R. We will also develop a framework for understanding converging lines of evidence for involvement of P2X4Rs expressed on macrophages in peripheral inflammatory pain.     

Cell-specific behavior of P2X7 receptors in mouse parotid acinar and duct cells

P2X7 receptors (P2X7Rs) affect many epithelial cell functions including transcellular ion transport, secretion, and cell death. Here we used parotid acinar and duct cells to reveal the unique cell-specific assembly and gating of the P2X7R channels. Immunolocalization indicated expression of P2X7Rs in the luminal membrane of both cell types. Stimulation with 5 mm ATP raised [Ca2+]i levels in a cell-specific manner and activated multiple currents. The current mediated by P2X7R was isolated by infusing the cells with high [EGTA]. The initial activation of acinar cell P2X7Rs by ATP was slow requiring approximately 2.5 min. Subsequent removal and addition of ATP, however, resulted in rapid inhibition and activation (gating) of the P2X7Rs. By contrast, P2X7Rs in duct cells displayed only rapid gating by ATP. Activation of P2X7Rs in both cell types was verified by (a) low Km for ATP, (b) sensitivity to external divalent ions, (c) lack of desensitization/inactivation, (d) permeability to Na+, and (e) inhibition by Brilliant Blue G, Cu2+, and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium. The slow P2X7R activation in acinar cells was not affected by manipulation of exo-/endocytosis. Rather, disassembly or solidification of the actin cytoskeleton prior to incubation with ATP prevented channel assembly. Remarkably, after completion of the slow activation, manipulation of the actin cytoskeleton no longer affected gating by ATP. Accordingly, manipulation of the actin cytoskeleton had no effect on P2X7R gating by ATP in duct cells. We concluded that P2X7Rs are not active in resting acinar cells. On exposure to ATP, P2X7Rs are assembled into functional channels with the aid of the actin cytoskeleton. Once assembled, P2X7Rs are subject to rapid gating by ATP. Duct cell P2X7Rs are preassembled and therefore continually subject to rapid gating by ATP. This cell-specific behavior may reflect the specific function of P2X7Rs in the two cell types.     

Alcohol action on membrane ion channels gated by extracellular ATP (P2X receptors).

Extracellular adenosine 5'-triphosphate (ATP) has been reported to produce excitatory actions in the nervous system, such as excitatory postsynaptic potentials or currents in both central and peripheral neurons, via activation of a class of ATP-gated membrane ion channels designated P2X receptors. This article reviews studies of alcohol effects on these receptor-channels. Ethanol has been found to inhibit ATP-gated ion channel function by shifting the agonist concentration-response curve to the right in a parallel manner, increasing the EC50 without affecting Emax of this curve. To distinguish whether this inhibition involves competitive antagonism of agonist action or a decrease in the affinity of the agonist binding site, the kinetics of activation and deactivation of agonist-activated current were studied. Ethanol was found to decrease the time-constant of deactivation of ATP-gated ion channels without affecting the time-constant of activation, indicating that ethanol inhibits the function of these receptors by an allosteric decrease in the affinity of the agonist binding site. The inhibition of ATP-gated ion channel function by a number of alcohols was found to exhibit a distinct cutoff effect that appeared to be related to the molecular volume of the alcohols. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. This cutoff effect has been interpreted as evidence that alcohols inhibit the function of ATP-gated ion channels by interacting with a hydrophobic pocket of circumscribed dimensions on the receptor protein. To evaluate the localization of this presumed alcohol binding site, the effect of the intracellular application of ethanol was studied on the inhibition of ATP-activated current by extracellularly applied ethanol. The intracellular application of 100 mM ethanol did not affect the inhibition of current by 100 mM extracellular ethanol, suggesting that the alcohol inhibition of ATP-gated ion channel function involves the extracellular domain of the receptor. Finally, recent studies suggest that the alcohol sensitivity of ATP-gated channels may be regulated by physiological mechanisms.     

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca²⁺ responses to ATP, including a transition of Ca²⁺ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10⁻⁴ and 10⁻² M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP₃) mediated Ca²⁺ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca²⁺ responses due to the biphasic nature of IP₃Rs and the interaction of SERCA pumps with IP₃Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP₃Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca²⁺ response signatures, which are important in mediating bone mechanoadaptation.     

Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells

The P2X4 receptor (P2X4R) is a member of a family of ATP-gated cation channels that are composed of three subunits. Each subunit has two transmembrane (TM) domains linked by a large extracellular loop and intracellularly located N- and C-termini. The receptors are expressed in excitable and non-excitable cells and have been implicated in the modulation of membrane excitability, calcium signaling, neurotransmitter and hormone release, and pain physiology. P2X4Rs activate rapidly and desensitize within the seconds of agonist application, both with the rates dependent on ATP concentrations, and deactivate rapidly and independently of ATP concentration. Disruption of conserved cysteine ectodomain residues affects ATP binding and gating. Several ectodomain residues of P2X4R were identified as critical for ATP binding, including K67, K313, and R295. Ectodomain residues also account for the allosteric regulation of P2X4R; H140 is responsible for copper binding and H286 regulates receptor functions with protons. Ivermectin sensitized receptors, amplified the current amplitude, and slowed receptor deactivation by binding in the TM region. Scanning mutagenesis of TMs revealed the helical topology of both domains, and suggested that receptor function is critically dependent on the conserved Y42 residue. In this brief article, we summarize this study and re-interpret it using a model based on crystallization of the zebrafish P2X4.1 receptor.     

Contribution of P2X4 Receptors to Ethanol Intake in Male C57BL/6 Mice

P2X receptors (P2XRs) are a family of cation-permeable ligand-gated ion channels activated by synaptically released extracellular adenosine 5′-triphosphate. The P2X4 subtype is abundantly expressed in the central nervous system and is sensitive to low intoxicating ethanol concentrations. Genetic meta-analyses identified the p2rx4 gene as a candidate gene for innate alcohol intake and/or preference. The current study used mice lacking the p2rx4 gene (knockout, KO) and wildtype (WT) C57BL/6 controls to test the hypothesis that P2X4Rs contribute to ethanol intake. The early acquisition and early maintenance phases of ethanol intake were measured with three different drinking procedures. Further, we tested the effects of ivermectin (IVM), a drug previously shown to reduce ethanol’s effects on P2X4Rs and to reduce ethanol intake and preference, for its ability to differentially alter stable ethanol intake in KO and WT mice. Depending on the procedure and the concentration of the ethanol solution, ethanol intake was transiently increased in P2X4R KO versus WT mice during the acquisition of 24-h and limited access ethanol intake. IVM significantly reduced ethanol intake in P2X4R KO and WT mice, but the degree of reduction was 50 % less in the P2X4R KO mice. Western blot analysis identified significant changes in γ-aminobutyric acid_A receptor α1 subunit expression in brain regions associated with the regulation of ethanol behaviors in P2X4R KO mice. These findings add to evidence that P2X4Rs contribute to ethanol intake and indicate that there is a complex interaction between P2X4Rs, ethanol, and other neurotransmitter receptor systems.     

A protein kinase C site highly conserved in P2X subunits controls the desensitization kinetics of P2X(2) ATP-gated channels

P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP receptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and voltage clamp recording in Xenopus oocytes, we identified the highly conserved protein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (time constant, >1 min) rat P2X(2) receptors. Mutant receptors P2X(2)T18A, T18N, and K20T devoid of this consensus site exhibited quickly desensitizing properties (time constant, <1 s). In contrast with wild-type receptors, mutant P2X(2) receptors with truncated C terminus exhibited variable cell-specific kinetics with quickly desensitizing currents converted to slowly desensitizing currents by phorbol ester-mediated stimulation of protein kinase C. Phosphorylation of Thr(18) was demonstrated directly by immunodetection using specific monoclonal antibodies directed against the phosphothreonine-proline motif. Our data indicate that both phosphorylation of the conserved threonine residue in the N-terminal domain by protein kinase C and interaction between the two cytoplasmic domains of P2X(2) subunits are necessary for the full expression of slowly desensitizing ATP-gated channels.     

An antagonist-insensitive P2X receptor expressed in epithelia and brain.

A cDNA was cloned which encodes a new ATP-gated ion channel (P2X4 receptor). ATP induces a cationic current in HEK293 cells transfected with the P2X4 receptor. However, the current is almost completely insensitive to antagonists effective at other P2X receptors. Sensitivity to two of these antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and pyridoxal 5-phosphate) is restored by replacement of Glu249 by lysine, which occurs at the equivalent position in P2X1 and P2X2 receptors. P2X4 RNA is found by in situ hybridization in the brain, peripheral ganglia and epithelia including serosal cells of salivary glands. Recordings from rat submandibular gland cells showed ATP-induced currents that are also insensitive to antagonists. These results define a further member of P2X receptor family, and they identify an amino acid residue involved in antagonist binding. They also introduce a new phenotype for ATP responses at P2X receptors--insensitivity to currently known antagonists.     

Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit

The P2X(3) receptor is an ATP-gated ion channel predominantly expressed in nociceptive neurons from the dorsal root ganglion. P2X(3) receptor channels are highly expressed in sensory neurons and probably contribute to the sensation of pain. Kinetics of P2X(3) currents are characterized by rapid desensitization (<100 ms) and slow recovery (>20 s). Thus, any mechanism modulating rate of desensitization and/or recovery may have profound effect on susceptibility of nociceptive neurons expressing P2X(3) to ATP. Here we show that currents mediated by P2X(3) receptor channels and the heteromeric channel P2X(2/3) composed of P2X(2) and P2X(3) subunits are potentiated by the neuropeptides substance P and bradykinin, which are known to modulate pain perception. The effect is mediated by the respective neuropeptide receptors, can be mimicked by phorbol ester and blocked by inhibitors of protein kinases. Together with data from site-directed mutagenesis our results suggest that inflammatory mediators sensitize nociceptors through phosphorylation of P2X(3) and P2X(2/3) ion channels or associated proteins.     

(+)-Anatoxin-a Is a Potent Agonist at Neuronal Nicotinic Acetylcholine Receptors

Abstract: The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC₅₀= 48 n M ) in stimulating ⁸⁶Rb⁺ influx into M10 cells, which express the nicotinic receptor subtype comprising α4 and β2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC₅₀= 140 n M ). α-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC₅₀ of 3.9 γ M , whereas α7 homooligomers reconstituted in Xenopus oocytes yielded an EC₅₀ value of 0.58 γ M for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (–)-nicotine and ˜20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.     

Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP: mapping agonist binding and channel gating

P2X receptors are ATP-gated cation channels. The x-ray structure of a P2X4 receptor provided a major advance in understanding the molecular basis of receptor properties. However, how agonists are coordinated, the extent of the binding site, and the contribution of the vestibules in the extracellular domain to ionic permeation have not been addressed. We have used cysteine-scanning mutagenesis to determine the contribution of residues Glu(52)-Gly(96) to human P2X1 receptor properties. ATP potency was reduced for the mutants K68C, K70C, and F92C. The efficacy of the partial agonist BzATP was also reduced for several mutants forming the back of the proposed agonist binding site. Molecular docking in silico of both ATP and BzATP provided models of the agonist binding site consistent with these data. Individual cysteine mutants had no effect or slightly increased antagonism by suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate. Mutants at the entrance to and lining the upper vestibule were unaffected by cysteine-reactive methanethiosulfonate (MTS) reagents, suggesting that it does not contribute to ionic permeation. Mutants that were sensitive to modification by MTS reagents were predominantly found either around the proposed ATP binding pocket or on the strands connecting the binding pocket to the transmembrane region and lining the central vestibule. In particular, ATP sensitivity and currents were increased by a positively charged MTS reagent at the G60C mutant at the interface between the central and extracellular vestibule. This suggests that dilation of the base of the central vestibule contributes to gating of the receptor.     

Lipid Raft Association and Cholesterol Sensitivity of P2X1-4 Receptors for ATP: CHIMERAS AND POINT MUTANTS IDENTIFY INTRACELLULAR AMINO-TERMINAL RESIDUES INVOLVED IN LIPID REGULATION OF P2X1 RECEPTORS*

Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20–23 and 27–29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.     

Roles of individual N-glycans for ATP potency and expression of the rat P2X1 receptor

P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X(1) receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn(284) remains unused because of a proline in the +4 position. The four other sites (Asn(153), Asn(184), Asn(210), and Asn(300)) carry N-glycans, but solely Asn(300) located only eight residues upstream of the predicted reentry loop of P2X(1) acquires complex-type carbohydrates. Like parent P2X(1), glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) diminishes or increases functional expression levels, respectively. In addition, elimination of Asn(210) causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X(1) receptors is severely impaired or abolished, respectively. We conclude that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X(1) receptors and that individual N-glycans possess marked positional effects on expression levels (Asn(154), Asn(210)) and ATP potency (Asn(210)).     

A “Septide-Sensitive” Receptor Is Not Involved in Tachykinin-Mediated Secretory and Inositol Phosphate Responses in Rat Parotid Gland: Are Several Transduction Pathways Involved After the Stimulation of the NK1 Receptor?

Abstract: In the rat parotid gland, the neuropeptide substance P (SP), as well as SP(4–11), and septide elicited inositol phosphate production (EC₅₀ values 0.44, 2, and 20 n M , respectively). No additivity of the maximal response to the three agonists was observed. SP, SP(4–11), and septide also stimulated protein secretion; for SP, two EC₅₀ were determined (0.5 and 160 n M ), whereas a single one could be determined for SP(4–11) and septide (EC₅₀ values 15 and 20 n M , respectively). The selective tachykinin NK₁ receptor antagonist RP67580 acted as a competitive inhibitor of both SP- and SP(4–11)-induced inositol phosphate production. Its effect on septide-induced inositol phosphate production was noncompetitive. RP67580 is apparently as potent at antagonizing septide, SP, or SP(4–11) (in all cases K _B = 3 n M ). These results show that in parotid gland, only NK₁ receptors are activated by SP, SP(4–11), and septide. We also showed that the protein secretion stimulated by SP was inhibited competitively by RP67580, whereas the effect of RP67580 was noncompetitive on protein secretion when SP(4–11) or septide was used. Our data indicate that in rat parotid gland, the existence of a specific "septide-sensitive" receptor can be ruled out and that only the NK₁ receptor is present and mediates cellular responses. Taken together, these results show that in this tissue the NK₁ receptor would present at least two different binding sites that could be coupled to different transduction pathways and that would regulate protein secretion.     

Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor

The transition from the closed to open state greatly alters the intra- and inter-subunit interactions of the P2X receptor (P2XR). The interactions that occur in the transmembrane domain of the P2X2R remain unclear. We used substituted cysteine mutagenesis disulfide mapping to identify pairs of residues that are in close proximity within the transmembrane domain of rP2X2R and compared our results to the predicted positions of these amino acids obtained from a rat P2X2R homology model of the available open and closed zebrafish P2X4R structures. Alternations in channel function were measured as a change in the ATP-gated current before and after exposure to dithiothreitol. Thirty-six pairs of double mutants of rP2X2R expressed in HEK293 cells produced normal functioning channels. Thirty-five pairs of these mutants did not exhibit a functionally detectable disulfide bond. The double mutant H33C/S345C formed redox-dependent cross-links in the absence of ATP. Dithiothreitol ruptured the disulfide bond of H33C/S345C and induced a 2 to 3-fold increase in current. The EC₅₀ for H33C/S345C before dithiothreitol treatment was ∼2-fold higher than that after dithiothreitol treatment. Dithiothreitol reduced the EC₅₀ to wild-type levels. Furthermore, expression of trimeric concatamer receptors with Cys mutations at some but not all six positions showed that the more disulfide bond formation sites within the concatamer, the greater current potentiation after dithiothreitol incubation. Immunoblot analysis of H33C/S345C revealed one monomer band under nonreducing conditions strongly suggesting that disulfide bonds are formed within single subunits (intra-subunit) and not between two subunits (inter-subunit). Taken together, these data indicate that His33 and Ser345 are proximal to each other across an intra-subunit interface. The relative movement between the first transmembrane and the second transmembrane in the intra-subunit is likely important for transmitting the action of ATP binding to the opening of the channel.     

Dual Gating Mechanism and Function of P2X7 Receptor Channels

The ATP-gated P2X7 receptor channel (P2X7R) operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.