Fig. 4. cAMP increases membrane turnover in HT29-CL19A cells, but this effect is unrelated to CFTR activation or syntaxin 1A regulation. (A) Representative simultaneous recordings of Gm, Cm and FM1-43 intensity in HT29-CL19A cells. Disruption of the syntaxin 1A-CFTR interaction with GST-Syn1AC results in a large increase in Gm. Note that while FM1-43 intensity increases when Gm increases in response to cAMP activation, there is no further increase in intensity when GST-Syn1AC disrupts the syntaxin-1 A-CFTR interaction. Also, whereas the addition of -Dyn results in a large increase in C_m in the presence of GST-Syn1AC, G_m did not change, indicating that inhibition of endocytosis did not augment conductance. Changes induced by GST-Syn1AC were similar to those induced by GST-Munc 18 (data not shown). (B) A summary of the changes in G_m, C_m and FM1-43 fluorescent intensity in HT29-CL19A cells on stimulation of current with cAMP under the various conditions indicated. When CFTR was activated by cAMP, a G_m of 0.73±0.23 nS (n=4) was measured. Dissociation of the syntaxin 1A-CFTR interaction by either GST-Syn1AC or GST-Munc 18 resulted in an increase in G_m to 1.55±0.19 nS (n=4) or 1.41±0.16 nS (n=5) respectively, while conductance remained unchanged in the presence of GST-Syn1AH3 [G_m=0.53±0.08 nS (n=5))]. In the HT29-CL19A cells, fluorescence intensity increased to the same degree upon activation of I_CFTR regardless of whether or not the syntaxin-1A-CFTR interaction was interrupted (range of increases was 11.6±0.75% to 17.2±4.2% (n=4-7), no significant differences were detected on comparison of these groups). C_m did not change unless endocytosis was inhibited by -Dyn. Measured capacitance in the absence of inhibition of endocytosis was consistently less than 0.075 pF. In the presence of -Dyn, capacitance increased to 1.16±0.26 pF (n=7) and 1.27±0.32 pF (n=5) when interrupting the protein-protein interactions with GST-Syn1AC and GST-Munc18, respectively. 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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