Identification of a distinct desensitisation gate in the ATP-gated P2X2 receptor.

P2X receptors are trimeric ATP-gated ion channels. In response to ATP binding, conformational changes lead to opening of the channel and ion flow. Current flow can decline during continued ATP binding in a process called desensitisation. The rate and extent of desensitisation is affected by multiple factors, for instance the T18A mutation in P2X2 makes the ion channel fast desensitising. We have used this mutation to investigate whether the gate restricting ion flow is different in the desensitised and the closed state, by combining molecular modelling and cysteine modification using MTSET (2-(Trimethylammonium)ethyl methanethiosulfonate). Homology modelling of the P2X2 receptor and negative space imaging of the channel suggested a movement of the restriction gate with residue T335 being solvent accessible in the desensitised, but not the closed state. This was confirmed experimentally by probing the accessibility of T335C in the P2X2 T18A/T335C (fast desensitisation) and T335C (slow desensitisation) mutants with MTSET which demonstrates that the barrier to ion flow is different in the closed and the desensitised states. To investigate the T18A induced switch in desensitisation we compared molecular dynamics simulations of the wild type and T18A P2X2 receptor which suggest that the differences in time course of desensitisation are due to structural destabilization of a hydrogen bond network of conserved residues in the proximity of T18.

Organization of ATP-gated P2X1 receptor intracellular termini in apo and desensitized states.

The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating-for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.

Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvß6 Expression.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFß1 is considered central to the pathogenesis of IPF. A major mechanism of TGFß1 activation in the lung involves the epithelially restricted αvß6 integrin. Expression of the αvß6 integrin is dramatically increased in IPF. How αvß6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the ß6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvß6 integrin expression in vitro We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvß6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

Influenza promotes collagen deposition via αvß6 integrin-mediated transforming growth factor ß activation.

Influenza infection exacerbates chronic pulmonary diseases, including idiopathic pulmonary fibrosis. A central pathway in the pathogenesis of idiopathic pulmonary fibrosis is epithelial injury leading to activation of transforming growth factor ß (TGFß). The mechanism and functional consequences of influenza-induced activation of epithelial TGFß are unclear. Influenza stimulates toll-like receptor 3 (TLR3), which can increase RhoA activity, a key event prior to activation of TGFß by the αvß6 integrin. We hypothesized that influenza would stimulate TLR3 leading to activation of latent TGFß via αvß6 integrin in epithelial cells. Using H1152 (IC50 6.1 µm) to inhibit Rho kinase and 6.3G9 to inhibit αvß6 integrins, we demonstrate their involvement in influenza (A/PR/8/34 H1N1) and poly(I:C)-induced TGFß activation. We confirm the involvement of TLR3 in this process using chloroquine (IC50 11.9 µm) and a dominant negative TLR3 construct (pZERO-hTLR3). Examination of lungs from influenza-infected mice revealed augmented levels of collagen deposition, phosphorylated Smad2/3, αvß6 integrin, and apoptotic cells. Finally, we demonstrate that αvß6 integrin-mediated TGFß activity following influenza infection promotes epithelial cell death in vitro and enhanced collagen deposition in vivo and that this response is diminished in Smad3 knock-out mice. These data show that H1N1 and poly(I:C) can induce αvß6 integrin-dependent TGFß activity in epithelial cells via stimulation of TLR3 and suggest a novel mechanism by which influenza infection may promote collagen deposition in fibrotic lung disease.