Extracellular histidine residues identify common structural determinants in the copper/zinc P2X2 receptor modulation

To assess the mechanism of P2X2 receptor modulation by transition metals, the cDNA for the wild-type receptor was injected to Xenopus laevis oocytes and examined 48-72 h later by the two-electrode voltage-clamp technique. Copper was the most potent of the trace metals examined; at 10 microm it evoked a 25-fold potentiation of the 10 microm ATP-gated currents. Zinc, nickel or mercury required 10-fold larger concentrations to cause comparable potentiations, while palladium, cobalt or cadmium averaged only 12- and 3-fold potentiations, respectively. Platinum was inactive. The non-additive effect of copper and zinc at 10-100 microm suggests a common site of action; these metals also shifted to the left the ATP concentration-response curves. To define residues necessary for trace metal modulation, alanines were singly substituted for each of the nine histidines in the extracellular domain of the rat P2X2 receptor. The H120A and H213A mutants were resistant to the modulator action of copper, zinc and other metals with the exception of mercury. Mutant H192A showed a reduction but not an abrogation of the copper or zinc potentiation. H245A showed less affinity for copper while this mutant flattened the zinc-induced potentiation. Mutant H319A reduced the copper but not the zinc-induced potentiation. In contrast, mutants H125A, H146A, H152A and H174A conserved the wild-type receptor sensitivity to trace metal modulation. We propose that His120, His192, His213 and His245 form part of a common allosteric metal-binding site of the P2X2 receptor, which for the specific coordination of copper, but not zinc, additionally involves His319.     

A histidine scan to probe the flexibility of the rat P2X2 receptor zinc-binding site

The response of P2X(2) receptors to submaximal concentrations of ATP is potentiated by low levels of extracellular zinc. Histidines 120 and 213 have previously been shown to be essential in binding zinc across an intersubunit binding site. We tested the flexibility of the zinc-binding site by making mutations that had the effect of shifting the two essential histidines up to 13 residues upstream or downstream from their original positions and then testing the ability of the mutated receptors to respond to zinc. Using this method, we were able to explore potential orientations of the two regions relative to one another. Our data are consistent with a moderately flexible zinc-binding site and inconsistent with parallel and anti-parallel orientations of the regions surrounding histidines 120 and 213.     

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.     

Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc

To elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site.     

Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptor channel: critical roles of CYS132 for zinc potentiation and ASP138 for copper inhibition

Zinc and copper are atypical modulators of ligand-gated ionic channels in the central nervous system. We sought to identify the amino acids of the rat P2X4 receptor involved in trace metal interaction, specifically in the immediate linear vicinity of His140, a residue previously identified as being critical for copper-induced inhibition of the ATP-evoked currents. Site-directed mutagenesis replaced conspicuous amino acids located within the extracellular domain region between Thr123 and Thr146 for alanines. cDNAs for the wild-type and the receptor mutants were expressed in Xenopus laevis oocytes and examined by the two-electrode technique. Cys132, but not Cys126, proved crucial for zinc-induced potentiation of the receptor activity, but not for copper-induced inhibition. Zinc inhibited in a concentration-dependent manner the ATP-gated currents of the C132A mutant. Likewise, Asp138, but not Asp131 was critical for copper and zinc inhibition; moreover, mutant D138A was 20-fold more reactive to zinc potentiation than wild-type receptors. Asp129, Asp131, and Thr133 had minor roles in metal modulation. We conclude that this region of the P2X4 receptor has a pocket for trace metal coordination with two distinct and separate facilitator and inhibitor metal allosteric sites. In addition, Cys132 does not seem to participate exclusively as a structural receptor channel folding motif but plays a role as a ligand for zinc modulation highlighting the role of trace metals in neuronal excitability.     

Zinc Potentiation of the Glycine Receptor Chloride Channel Is Mediated by Allosteric Pathways

Abstract: Molecular mechanisms of zinc potentiation were investigated in recombinant human α1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [³H]strychnine binding assays. In the wild-type (WT) GlyR, 1 µ M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1–M2 and M2–M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2–M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.     

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor (GlyR) alpha1 subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha1 subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His107 from one subunit and His109 from an adjacent subunit. This was tested by co-expressing alpha1 subunits containing the H107A mutation with alpha1 subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha1 homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha1 subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha1beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha1 homomers. The binding of zinc at the interface between adjacent alpha1 subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.     

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.     

Covalent Modification of Mutant Rat P2X2 Receptors with a Thiol-Reactive Fluorophore Allows Channel Activation by Zinc or Acidic pH without ATP

Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C₅-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.     

Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K(D) = 10-30 nm) and low (K(D) = 0.1-1.0 microm) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat beta2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type alpha1 and gamma2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [(3)H]Ro15-4513 binding or impaired allosteric interactions between the low affinity GABA and benzodiazepine sites. Although mutations at position 74 had little effect on [(3)H]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [(3)H]muscimol binding sites by approximately 6-fold, and the Y62S mutation led to a loss of detectable high affinity binding sites. After expression in oocytes, the EC(50) values for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude that Tyr-62 of the beta subunit is an important determinant for high affinity agonist binding to the GABA(A) receptor.     

Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor

The P2X7 receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X7 receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 micromol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X7 receptor allosteric modulation.     

Ribosomal Intersubunit Bridge B2a Is Involved in Factor-Dependent Translation Initiation and Translational Processivity

Intersubunit bridges are important for holding together subunits in the 70S ribosome. Moreover, a number of intersubunit bridges have a role in modulating the activity of the ribosome during translation. Ribosomal intersubunit bridge B2a is formed by the interaction between the conserved 23S rRNA helix-loop 69 (H69) and the top of the 16S rRNA helix 44. Within the 70S ribosome, bridge B2a contacts translation factors and the A-site tRNA. In addition to bridging the subunits, bridge B2a has been invoked in a number of other ribosomal functions from initiation to termination. In the present work, single-nucleotide substitutions were inserted at positions 1912 and 1919 of Escherichia coli 23S rRNA (helix 69), which are involved in important intrahelical and intersubunit tertiary interactions in bridge B2a. The resulting ribosomes had a severely reduced activity in a cell-free translation elongation assay, but displayed a nearly wild-type-level peptidyl transferase activity. In vitro reassociation efficiency decreased with all of the H69 variant 50S subunits, but was severest with the A1919C and ΔH69 variants. The mutations strongly affected initiation-factor-dependent 70S initiation complex formation, but exhibited a minor effect on the nonenzymatic initiation process. The mutations decreased ribosomal processivity in vitro and caused a progressive depletion of 50S subunits in polysomal fractions in vivo. Mutations at position 1919 decreased the stability of a dipeptidyl-tRNA in the A-site, whereas the binding of the dipeptidyl-tRNA was rendered more stable with 1912 and ΔH69 mutations. Our results suggest that the H69 of 23S rRNA functions as a control element during enzymatic steps of translation.     

Physiological Concentrations of Zinc Have Dual Effects on P2X Myenteric Receptors of Guinea Pig

We, hereby, characterize the pharmacological effects of physiological concentrations of Zinc on native myenteric P2X receptors from guinea-pig small intestine and on P2X2 isoforms present in most myenteric neurons. This is the first study describing opposite effects of Zinc on these P2X receptors. It was not possible to determine whether both effects were concentration dependent, yet the inhibitory effect was mediated by competitive antagonism and was concentration dependent. The potentiating effect appears to be mediated by allosteric changes induced by Zinc on P2X myenteric channels, which is more frequently observed in myenteric neurons with low zinc concentrations. In P2X2-1 and P2X2-2 variants, the inhibitory effect is more common than in P2X myenteric channels. However, in the variants, the potentiatory effect is of equal magnitude as the inhibitory effect. Inhibitory and potentiatory effects are likely mediated by different binding sites that appear to be present on both P2X2 variants. In conclusion, in myenteric native P2X receptors, Zinc has quantitatively different pharmacological effects compared to those observed on homomeric channels: P2X2-1 and P2X2-2. Potentiatory and inhibitory Zinc effects upon these receptors are mediated by two different binding sites. All our data suggest that myenteric P2X receptors have a more complex pharmacology than those of the recombinant P2X2 receptors, which is likely related to other subunits known to be expressed in myenteric neurons. Because these dual effects occur at Zinc physiological concentrations, we suggest that they could be involved in physiological and pathological processes.     

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.     

Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.     

Disulfide bond formation in the herpes simplex virus 1 UL6 protein is required for portal ring formation and genome encapsidation

The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol. 81:8868-8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.     

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels lacking the N-terminal domain

Ionotropic glutamate receptor (iGluR) subunits contain a approximately 400-residue extracellular N-terminal domain ("X domain"), which is sequence-related to bacterial amino acid-binding proteins and to class C G-protein-coupled receptors. The X domain has been implicated in the assembly, transport to the cell surface, allosteric ligand binding, and desensitization in various members of the iGluR family, but its actual role in these events is poorly characterized. We have studied the properties of homomeric alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-selective GluR-D glutamate receptors carrying N-terminal deletions. Our analysis indicates that, surprisingly, transport to the cell surface, ligand binding properties, agonist-triggered channel activation, rapid desensitization, and allosteric potentiation by cyclothiazide can occur normally in the complete absence of the X domain (residues 22-402). The relatively intact ligand-gated channel function of a homomeric AMPA receptor in the absence of the X domain indirectly suggests more subtle roles for this domain in AMPA receptors, e.g. in the assembly of heteromeric receptors and in synaptic protein interactions.     

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca²⁺ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca²⁺− and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca²⁺-independent basal activity and Ca²⁺-dependent activation of [³H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca²⁺ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca²⁺ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.     

High potency zinc modulation of human P2X2 receptors and low potency zinc modulation of rat P2X2 receptors share a common molecular mechanism

Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2–100 μm, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μm. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nm) to very low (>100 μm) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.     

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.