The Second Transmembrane Domain of P2X7 Contributes to Dilated Pore Formation

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors. © 2013 Sun et al

Rhinovirus induction of fractalkine (CX3CL1) in airway and peripheral blood mononuclear cells in asthma

Rhinovirus infection is associated with the majority of asthma exacerbations. The role of fractalkine in anti-viral (type 1) and pathogenic (type 2) responses to rhinovirus infection in allergic asthma is unknown. To determine whether (1) fractalkine is produced in airway cells and in peripheral blood leucocytes, (2) rhinovirus infection increases production of fractalkine and (3) levels of fractalkine differ in asthmatic compared to non-asthmatic subjects. Fractalkine protein and mRNA levels were measured in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) from non-asthmatic controls (n = 15) and mild allergic asthmatic (n = 15) subjects. Protein levels of fractalkine were also measured in macrophages polarised ex vivo to give M1 (type 1) and M2 (type 2) macrophages and in BAL fluid obtained from mild (n = 11) and moderate (n = 14) allergic asthmatic and non-asthmatic control (n = 10) subjects pre and post in vivo rhinovirus infection. BAL cells produced significantly greater levels of fractalkine than PBMCs. Rhinovirus infection increased production of fractalkine by BAL cells from non-asthmatic controls (P<0.01) and in M1-polarised macrophages (P<0.05), but not in BAL cells from mild asthmatics or in M2 polarised macrophages. Rhinovirus induced fractalkine in PBMCs from asthmatic (P<0.001) and healthy control subjects (P<0.05). Trends towards induction of fractalkine in moderate asthmatic subjects during in vivo rhinovirus infection failed to reach statistical significance. Fractalkine may be involved in both immunopathological and anti-viral immune responses to rhinovirus infection. Further investigation into how fractalkine is regulated across different cell types and into the effect of stimulation including rhinovirus infection is warranted to better understand the precise role of this unique dual adhesion factor and chemokine in immune cell recruitment