Paper Title
The Regulation of Calcineurin and CFTR as a Switch in Cell Secretion: A Functional Study

Abstract
Cystic fibrosis (CF) is a disease that affects respiratory function and in the UK it affects more than 151 young persons per 100,000 people. The disease arises due to dysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) protein, a protein that has been shown recently to influence calcineurin activities in cell secretion.CFTR dysfunction causing mucus lodging and bacteria colonisation of the airways and intestinal linings leading to functional alterations of immune cells. In the airways, CFTR has been shown to form a functional complex with S100A10 and Annexin A2 in a cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA) dependent manner. The multiprotein complex of CFTR, S100A10 and Annexin A2 is also regulated by protein phosphatase 2B (PP2B). The objectives of this study was to investigate whether chloride ion (Cl−) channels are activated by lipopolysaccharide (LPS) from Pseudomonas aeruginosa (PA), if this activation is mediated by cAMP/PKA/PP2B pathway and to investigate the role of Cl− channels in cytokines release by immune cells during PAinfection. PMA-stimulated monocytic THP-1 and primary human monocytes were used in the study. Whole cell patch records showed that LPS from PA can activate Cl− channels, and this activation appears to require an intact PKA/PP2B signalling pathway. The Gout in the presence of LPS was 2185.97± 226 μS/cm2 (n=27) and this was significantly suppressed by diisothiocyanatostilbene-disulfonic acid (DIDS), an outwardly-rectifying Cl− channel (ORCC) blocker to 1204.40±132 μS/cm2. The Goutwas further suppressed by CFTR inhibition by CFTRinh172 to 838.68±101 μS/cm2. Data from cells stimulated with LPS from PA that were pre-incubated with PKA inhibitor or PP2B inhibitor showed no DIDS and CFTRinh172 sensitive currents. Activation of both CFTR and ORCC was however, observed in response to exposure of monocytes to LPS. In addition, ELISA showed that the activation of CFTR and ORCC plays a role in mediating the release of pro-inflammatory cytokines such as IL-1β upon exposure of monocytes to LPS. However, this secretion was significantly inhibited due to CFTR and ORCC inhibition. In conclusion, our data confirmed that LPS from PA activates Cl− channels in PMA-stimulated monocytic THP-1 and primary human monocytes.Our dataalso confirmedthat Cl− channels were involved in IL-1β release in both cells upon exposure to LPS.However, it has been found that PKA does not seem to influence the Cl−dependent cytokine release. Keywords - Cystic Fibrosis, CFTR, Camp, Chloride Ion Channels, Lipopolysaccharide, IL-1β, Immune Cells