Lin-Hua Jiang

Proteomic and functional evidence for a P2X7 receptor signalling complex.

P2X receptors are ATP‐gated ion channels in the plasma membrane, but activation of the P2X7 receptor also leads to rapid cytoskeletal re‐arrangements such as membrane blebbing. We identified 11 proteins in human embryonic kidney cells that interact with the rat P2X7 receptor, by affinity purification followed by mass spectroscopy and immunoblotting [laminin α3, integrin β2, β‐actin, α‐actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4‐kinase and receptor protein tyrosine phosphatase‐β (RPTPβ)]. Activation of the P2X7 receptor resulted in its dephosphorylation. Whole‐cell recordings from cells expressing P2X7 receptors showed that this markedly reduced subsequent ionic currents and it also slowed membrane bleb formation. By mutagenesis, we identified Tyr343 in the putative second transmembrane domain as the site of phosphorylation. Thus, we have identified a P2X7 receptor signalling complex, some members of which may initiate cytoskeletal rearrangements following receptor activation. Others, such as RPTPβ, might exert feedback control of the channel itself through its dephosphorylation.

Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor.

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X2receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys69 and Lys71). We substituted cysteines in this region (Asp57 to Asp71) and found that for S65C and I67C ATP-evoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile67 contribute to the ATP-binding site.

TRPC channel activation by extracellular thioredoxin

Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5–TRPC1 heteromultimeric channels by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5–TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.

New structure enlivens interest in P2X receptors.

P2X receptors are ATP-gated membrane ion channels with multifarious roles, including afferent sensation, autocrine feedback loops, and inflammation. Their molecular operation has been less well elucidated compared with other ligand-gated channels (nicotinic acetylcholine receptors, ionotropic glutamate receptors). This will change with the recent publication of the crystal structure of a closed P2X receptor. Here we re-interpret results from 15 years of experiments using site-directed mutagenesis with a model based on the new structure. Previous predictions of receptor stoichiometry, the extracellular ATP binding site, inter-subunit contacts, and many details of the permeation pathway fall into place in three dimensions. We can therefore quickly understand how the channel operates at the molecular level. This is important not only for ion- channel aficionados, but also those engaged in developing effective antagonists at P2X receptors for potential therapeutic use.

Characterization of a selective and potent antagonist of human P2X7 receptors, AZ11645373

The ATP‐gated P2X7 receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti‐inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X7 receptor‐mediated dye uptake in human macrophage THP‐1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized.

Interactions, Functions, and Independence of Plasma Membrane STIM1 and TRPC1 in Vascular Smooth Muscle Cells

Stromal interaction molecule 1 (STIM1) is a predicted single membrane–spanning protein involved in store-operated calcium entry and interacting with ion channels including TRPC1. Here, we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a nonselective cationic current in response to store depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA. Calcium entry evoked by store depletion was partially inhibited by STIM1 short interfering RNA, whereas calcium release was unaffected. STIM1 short interfering RNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1, and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localization studies revealed TRPC1 alone, as well as with STIM1. The data suggest a complex situation in which there is not only plasma membrane–spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current but also TRPC1-STIM1 interaction, a TRPC1-dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation.

Role of ectodomain lysines in the subunits of the heteromeric P2X2/3 receptor.

Lysine residues near each end of the receptor ectodomain (in rat P2X2 Lys69 and Lys308) have been implicated in ATP binding to P2X receptors. We recorded membrane currents from human embryonic kidney cells expressing P2X subunits and found that lysine-to-alanine substitutions at equivalent positions in the P2X3 receptor (Lys63 and Lys299) also prevented channel function. Heteromeric P2X2/3 receptors are formed when P2X2 and P2X3 subunits are expressed together; they can be distinguished by their relatively sustained response to αβ-methylene-ATP. By coexpression of wild-type P2X3 and mutated P2X2 subunit, we found that the heteromeric P2X2/3 channel functioned normally when either lysine in the P2X2 subunit was mutated to alanine (i.e., [K69A] or [K308A]) but not when both lysines were mutated to alanine (i.e., [K69A, K308A]). However, coexpression of wild-type P2X2 with a mutated P2X3 subunit ([K68A] or [K299A]) produced no functional heteromers. The rescue of the single lysine mutant P2X2 subunit by wild-type P2X3 (but not the converse) suggests that the heteromeric channel contains one P2X2 and two P2X3 subunits and that the receptor functions essentially normally as long as two subunits are not mutated. The failure to rescue function in the P2X2 subunit with both lysines mutated by wild-type P2X3 suggests that these residues from two different subunits interact in agonist binding or channel opening.

Robotic multiwell planar patch-clamp for native and primary mammalian cells

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4–8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1–6 h depending on the experimental design and yields 16–33 cell recordings.

Single nucleotide polymorphisms that were identified in affective mood disorders affect ATP-activated P2X7 receptor functions.

Genetic linkage studies have previously identified many single non-synonymous nucleotide polymorphisms (SNPs) in the human P2RX7 gene in individuals with affective mood disorders. The P2RX7 gene encodes the P2X(7) receptor (P2X(7)R) that operates as an ATP-activated Ca(2+)-permeable cationic channel and induces formation of a large pore, the two functional properties that are critical for the physiological and pathological roles of the receptor. The current knowledge regarding the effects of SNPs on the P2X(7)R functional properties, which is indispensable to help elucidate the disease mechanism, is limited. In this study, we introduced by site-directed mutagenesis twelve SNP mutations in the human P2X(7) receptor that were previously identified in or associated with affective mood disorders, expressed the resultant mutants in human embryonic kidney cells, and characterized their functional properties by electrophysiology. All mutations except Q460R gave rise to profound effects on the P2X(7)R function. G150R, E186K and I568N conferred complete loss of function. V76A, R117W, L191P, T357S and E496A resulted in strong impairment of, whereas H155Y and A348T caused significant increase in, both ATP-activated ion channel function and pore formation. Q521H reduced the receptors sensitivity to extracellular Ca(2+) inhibition. An atomic structure model of the human P2X(7)R, based on the crystal structure of the zebrafish P2X(4) receptor, suggests that the SNP mutational effects may result from changes in subunit interaction, agonist binding and/or channel gating. These results provide essential knowledge for a better understanding of the relationships between human P2RX7 SNPs and associated pathologies as well as the receptor structure-function relationships.

Identification of Key Residues Coordinating Functional Inhibition of P2X7 Receptors by Zinc and Copper

P2X7 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 P2X7 receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X7 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 His62 or Asp197 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 P2X7 receptor, primarily involving combined interaction with His62 and Asp197 residues.