||Mucociliary clearance is a primary innate defense mechanism for mammalian airways. The airway surface liquid (ASL), generated by submucosal glands, is critical for the effectiveness of this clearance process. Many ion channels and transporters are involved in the regulation of ASL secretion in the submucosal gland serous cells, including the apical cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the basolateral potassium channels. My research project aims to better understand the molecular mechanism of the regulation of anion secretion in Calu-3 human airway epithelial cells, a model of serous cells which are a key player in liquid secretion and pathogenesis of cystic fibrosis in human airways. Previous works have demonstrated that CFTR is regulated by a compartmentalized cAMP/PKA signaling pathway involving G protein coupled receptors (GPCRs) and the downstream signaling molecules. However, the mechanisms of the colocalization remain largely elusive. Lipid rafts are proposed to act as a platform to concentrate signaling molecules and enhance signaling efficiency in several signaling pathways including GPCR-mediated signaling. Thus, the first part of my thesis study attempted to investigate the role of lipid rafts in the localized signaling from GPCRs to CFTR in Calu-3 cells. Using sucrose gradient centrifugation methods, we found that CFTR, along with Gαs, was associated with lipid rafts, and the association was disrupted by cholesterol depletion with methyl-β-cyclodextrin (MCD) treatment. Using short circuit current (Isc) as a readout of CFTR activity in airway or colonic epithelial cells, we showed that cholesterol depletion by MCD did not affect the Isc induced by several GPCR agonists, although it increased basal membrane permeability. Similar results were also obtained with a cholesterol biosynthesis inhibitor lovastatin and a cholesterol binding agent filipin. Furthermore, cholesterol depletion did not impair cAMP production elicited by the GPCR agonists. Our data suggested that GPCR-mediated signaling maintain their integrity after lipid raft disruption in epithelial cells and cast doubts on the role of lipid rafts as a signaling platform in GPCR-mediated signaling. The second part of my thesis study focused on studying the adenosine receptors-mediated PLC signaling in the regulation of anion secretion in Calu-3 cells. We found that mucosal adenosine-induced anion secretion, measured by the Isc was inhibited by the PLC-specific inhibitor U-73122. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance. The third part of my thesis study investigated the mechanosensitive gating of CFTR channels. CFTR protein belongs to ATP-binding cassette (ABC) transporter superfamily and the gating of the CFTR channel is subject to ATP hydrolysis and phosphorylation. Therefore, CFTR is traditionally viewed as an intracellular ligand-gated ion channel. We show that the CFTR channel is robustly activated by membrane stretch induced by negative pressures as small as 5 mmHg at the single-channel, cellular, and tissue levels. Stretch increased the product of channel number and open probability (NPo). Stretch activation of CFTR is an intrinsic property that is independent of Ca2+ and cAMP signaling. Our study has revealed an unexpected function of CFTR, in addition to its roles as a ligand-gated anion channel and as a regulator of other membrane transporters, and is the first to report a mechanosensitive anion channel with a clearly defined molecular identity. Given that CFTR is often found in mechanically dynamic environments, its mechanosensitivity has important physiological implications in epithelial ion transport and cell volume regulation.