Fig. 3. cAMP and syntaxin 1A stimulate CFTR without affecting membrane turnover in human airway epithelial cells. (A) Representative simultaneous recordings of Gm, Cm and FM1-43 fluorescence intensity under various conditions in human airway epithelial cells. Horizontal bars indicate recording conditions in each representative cell. Each vertical column depicts a representative set of records from a single cell. While there is a progressive increase in Gm over the baseline with cAMP activation and then with addition of GST-Syn1AC, inhibition of endocytosis by 1 µg of -Dyn had no additional effect on Gm. Activation of ICFTR in 16HBE14o- cells occurs without an apparent contribution from membrane trafficking as evidenced by a lack of change in either Cm or FM1-43 fluorescence intensity even when endocytosis is inhibited by -Dyn. Inclusion of GST-Syn1AC or GST-Munc 18 in the patch pipette resulted in similar increases in Gm, both in terms of magnitude and time course. (B) A summary of the changes in Gm, Cm and FM1-43 fluorescence intensity in 16HBE14o- cells on stimulation of current with cAMP under the various conditions indicated. When CFTR was activated by cAMP, a Gm of 1.03 (0.33 nS (n=4)) was measured. Dissociation of the Syn1A-CFTR interaction by GST-Syn1AC resulted in an approximately threefold increase in Gm to 3.57 (0.74 nS (n=10)). Gm increase by GST-Munc 18 was also approximately threefold to 2.94 (0.20 (n=5)). No change in conductance was seen in the presence of GST-Syn1AH3 [Gm=1.32 (0.18 nS (n=5))]. For the 16HBE14o- cells, in all instances, disruption of the syntaxin 1A-CFTR interaction did not result in changes in measures of capacitance or FM1-43 fluorescent intensity. The asterisk (^*) indicates P<0.05 for comparisons with baseline conditions (in the absence of cAMP stimulation). # indicates P<0.05 for comparisons with conditions in which the syntaxin 1A-CFTR interaction was not disrupted.

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