TMEM16A/anoctamin-1 has been identified as a protein with the classic properties of a Ca2+-activated chloride channel. Here, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and chemical cross-linking to assess the quaternary structure of the mouse TMEM16A(a) and TMEM16A(ac) splice variants as well as a genetically concatenated TMEM16A(a) homodimer. The constructs carried hexahistidyl (His) tags to allow for their purification using a nondenaturing metal affinity resin. Neither His-tagging nor head-to-tail concatenation of two copies of TMEM16A(a) noticeably affected Ca2+-induced measured macroscopic Cl− currents compared with the wild-type TMEM16A(a) channel. The digitonin-solubilized, nondenatured TMEM16A(a) protein migrated in the BN-PAGE gel as a homodimer, as judged by comparison with the concatenated TMEM16A(a) homodimer and channel proteins of known oligomeric structures (e.g. the voltage-gated Cl− channel CLC-1). Cross-linking with glutaraldehyde corroborated the homodimeric structure of TMEM16A(a). The TMEM16A(a) homodimer detected in Xenopus laevis oocytes and HEK 293 cells dissociated into monomers following denaturation with SDS, and reducing versus nonreducing SDS-PAGE provided no evidence for the presence of intersubunit disulfide bonds. Together, our data demonstrate that the Ca2+-activated chloride channel member TMEM16A shares an obligate homodimeric architecture with the hCLC-1 channel.
P2X 7 receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X 7 subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X 7 subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434 -595) in Xenopus laevis oocytes. P2X 7 channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only ϳ5% of the full-length P2X 7 receptor current. Co-assembly of channel core subunits with full-length P2X 7 subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X 7 receptor activity. Co-expression of the tail extension as a discrete protein increased ATPgated current amplitudes of P2X 7 channel cores 10 -20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X 7 receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X 7 channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X 7 subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409 -436 and 434 -494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X 7 channel gating sensitive to intracellular regulation. The P2X 7 receptor, an ATP-gated cation channel, is expressed predominantly in immune cells (such as macrophages and lymphocytes), glial cells, and epithelial cells (for recent reviews, see Refs. 1 and 2). Activation of the P2X 7 receptor has been implicated in pivotal inflammatory responses resulting from ATP-stimulated pro-inflammatory cytokine release (particularly of interleukin-1 and interleukin-18) through exosomes (3) during cell proliferation and apoptosis (1, 2). These functions of the P2X 7 receptor have been attributed to its unusual dual role as a classic ligand-gated channel for small cations and as a cytolytic pore (4, 5).Cloning of the P2X 7 receptor revealed a typical P2X subunit "core" structure, with a short cytoplasmic NH 2 -terminal tail and two transmembrane segments connected by a large N-glycosylated ectodomain. However, the P2X 7 receptor has a carboxyl tail extension that is 120 -200 amino acids longer than that of the other six P2X family members, P2X 1 -P2X 6 (4, 5). Because the cytolytic pore-forming ability of P2X 7 is not shared by the other P2X receptor subtypes, this function has been plausibly assigned to the long carboxyl tail. Indeed, truncation of most of the extra portion of the carboxyl tail of P2X 7 prevented cytolytic pore formation, apparently without...
Unlike NAC, ambroxol is able to not only inhibit acute mediator release from mast cells and leukocytes but also reduce immunomodulatory cytokine generation from basophils and may have beneficial effects in the treatment of allergic respiratory diseases.
P2X4 and P2X7 are members of the P2X receptor family, comprising seven isoforms (P2X1–P2X7) that form homo- and heterotrimeric non-specific cation channels gated by extracellular ATP. P2X4 and P2X7 are widely coexpressed, particularly in secretory epithelial cells and immune and inflammatory cells, and regulate inflammation and nociception. Although functional heteromerization has been established for P2X2 and P2X3 subunits expressed in sensory neurons, there are contradictory reports regarding a functional interaction between P2X4 and P2X7 subunits. To resolve this issue, we coexpressed P2X4 and P2X7 receptor subunits labeled with green (EGFP) and red (TagRFP) fluorescent proteins in Xenopus laevis oocytes and investigated a putative physical interaction between the fusion proteins by Förster resonance energy transfer (FRET). Coexpression of P2X4 and P2X7 subunits with EGFP and TagRFP located in the extracellular receptor domains led to significant FRET signals. Significant FRET signals were also measured between C-terminally fluorophore-labeled full-length P2X41-384 and C-terminally truncated fluorescent P2X71-408 subunits. We furthermore used the two-electrode voltage clamp technique to investigate whether human P2X4 and P2X7 receptors (hP2X4, hP2X7) functionally interact at the level of ATP-induced whole-cell currents. Concentration–response curves and effects of ivermectin (P2X4-potentiating drug) or BzATP (P2X7-specific agonist) were consistent with a model in which coexpressed hP2X4 and hP2X7 do not interact. Similarly, the effect of adding specific inhibitors of P2X4 (PSB-15417) or P2X7 (oATP, A438079) could be explained by a model in which only homomers exist, and that these are blocked by the respective antagonist. In conclusion, we show that P2X4 and P2X7 subunits can form heterotrimeric P2X4/P2X7 receptors. However, unlike observations for P2X2 and P2X3, coexpression of P2X4 and P2X7 subunits does not result in a novel electrophysiologically discriminable P2X receptor phenotype.
Some recent observations have indicated that cells other than mast cells, notably macrophages, may contain significant amounts of histamine. Using a histamine-specific radioimmunoassay, we found that human blood monocytes and lymphocytes contain about 0.05 pg histamine/cell. Various other cells, e.g. fibroblasts, colorectal tumor, kidney and ovarian cells, and murine bone marrow derived macrophages contained markedly less histamine (< 0.008 pg/cell). Ionophore A23187 (1 microM) released up to 50% of the total histamine from monocytes and lymphocytes. C5a caused a dose-dependent histamine release of up to 40% in monocytes and up to 20% in lymphocytes. Substance P induced a release only in cells of certain donors. Lipopolysaccharide, concanavalin A, and compound 48/80 had no effect. Culturing of the cells caused a loss of cellular histamine and its releasability. In view of the huge numbers of monocytes and lymphocytes in the blood, the histamine in these cells has to be taken into account under both physiological and pathophysiological conditions.
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