2D). ISO, which raises cellular cAMP levels and activates CFTR via ��-adrenergic receptor activation, was also ineffective at increasing ASL volume compared to air-exposed controls phase 3 (Fig. 2D). In contrast, ATP was effective in raising ASL volume via Ca2+-activated Cl? secretion following CS (Fig. 2D). Collectively, these data suggest that in vitro, CS diminished CFTR Cl? channel function. The effect of CS on CFTR channel number and open probability The magnitude of the CFTR contribution to transepithelial Cl? secretion reflects both the number of CFTR Cl? channels at the apical cell membrane and the activation state of these channels (open probability, Po). We, therefore, tested the effects of CS on each component. Studies of CFTR channels reconstituted in bilayers revealed that CS induced no change in channel Po (Fig.

3A). However, using immunofluorescence techniques, we found that virtually all immunodetectable native CFTR was lost from the apical membrane of HBECs after CS exposure (Fig. 3B). In contrast, ezrin-binding protein 50 (EPB50), which helps anchor CFTR to the cytoskeleton, remained in the apical membrane after CS exposure (31), and the position of the basolateral Na+/K+ ATPase was not altered (Fig. 3B). Figure 3. CFTR is lost from the plasma membrane during CS exposure. A) Single CFTR channel lipid bilayer recordings. All points in the histogram shown on the left and 2 min of CFTR single-channel recording on the right. Open (O) and closed (C) states are indicated. … To examine the effects of CS on surface CFTR in more detail, we performed 2 studies with heterologously expressed CFTR.

First, we overexpressed HA-tagged CFTR in HBECs with an adenoviral vector to better localize CFTR internalization after CS exposure (28). In adenoviral/CFTR-transduced HBECs, CFTR internalization was increased post-CS, as compared to air exposure (Fig. 3C). Second, to study CFTR removal from the plasma membrane, we used BHKCFTR cells with an extracellular HA tag (29). Intact cells were treated with CS or air and then probed with an anti-HA antibody to image and quantify surface CFTR levels. Disappearance of CFTR from the plasma membrane was visible by confocal microscopy after CS exposure (Fig. 3D), and cell surface CFTR levels, as measured by cell surface ELISA, were also decreased (Fig. 3E). As with our studies of airway epithelia, this diminution was reversible, and by 60 min after the start of CS exposure (1 h total), surface CFTR levels had returned toward normal levels (Fig. 3D, E). Pretreatment with brefeldin A, which inhibits trafficking from the ER to the Golgi apparatus, prevented CFTR recovery (Fig. 3E). Thus, we conclude that CS contains materials that promote the acute loss of CFTR channels from plasma Drug_discovery membranes.