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Mechanisms of CFTR regulation by syntaxin 1A and PKA

Steven Y. Chang^1,*, Anke Di^2,*, Anjaparavanda P. Naren³, H. Clive Palfrey², Kevin L. Kirk³ and Deborah J. Nelson^2,

¹ Department of Medicine, Section of Pulmonary and Critical Care Medicine, The University of Chicago Hospitals, 5841 S. Maryland Avenue, MC 6026, Chicago, IL 60637, USA
² Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, 947 East 58^th St, MC 0926, Chicago, IL 60637, USA
³ Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, 1918 University Blvd, MCLM 985, Birmingham, AL 35294, USA

View larger version (29K): [in a new window]   Fig. 1. Syntaxin 1A and CFTR physically and functionally interact in 16HBE14o- cells. (A) Syntaxin-1A-specific IgG (Naren et al., 2000) can coimmunoprecipitate CFTR from 16HBE14o- cells. (B) Representative whole-cell ICFTR in 16HBE14o- cells maximally stimulated by addition of cAMP cocktail to the external bath with voltage steps ranging from -110 to +110 mV in increments of 10 mV in the absence and presence of 350 nM GST-Syn1AC. (C) Current-voltage relationship of ICFTR from (B). The reversal potentials for the Cl- current in control conditions and in the presence of GST-Syn1AC, GST-Munc18 and GST-Syn1AH3 were -18±1.5 mV (n=6), -15±2.7 mV (n=7), -10.2±1.2mV (n=5) and -14±1.5 mV (n=5), respectively. (D) The ICFTR was similar irrespective of whether disruption of Syn1A-CFTR interaction was by GST-Syn1AC or by 300 nM GST-Munc18. Whole-cell ICFTR in the presence of 350 nM GST-Syn1AH3 was not significantly different from the control currents. Current density for control ICFTR was 29.6±10.0 pA/pF (n=6). Current densities in the presence of GST-Syn1AC and GST-Munc18 increased threefold to 85.0±12.3 pA/pF (n=7) and 88.4±17.9 pA/pF (n=5) respectively. When Syn1AH3 was included in the pipette solution, the current density was 26.9±7.4 pA/pF (n=5). Asterisk (*) indicates p <0.05 when comparing the densities of either control currents or ICFTR in the presence of GST-Syn1AH3 with currents in the presence of either GST-Syn1AC or GST-Munc 18.

View larger version (11K): [in a new window]   Fig. 2. Cytochalasin D prevents augmentation of ICFTR by the cytosolic domain of syntaxin 1A. Whole-cell currents were obtained from 16HBE4o- cells as in Fig. 1. A summary of current density at 110 mV is given for cells under control conditions and cells stimulated with the cAMP cocktail added to the bath solution as indicated under the respective bars. Cells were treated with 0.5 µg/ml of cytochalasin D for 30 minutes prior to whole-cell voltage clamp in the presence and absence of soluble syntaxin 1AC (20 µg/ml) added to the pipette solution. There was no significant difference between ICFTR in the presence and absence of the cytoskeleton-disrupting reagent. ICFTR in the presence of the cytosolic domain of syntaxin 1A was not significantly different from that observed under control conditions.

View larger version (20K): [in a new window]   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.

View larger version (21K): [in a new window]   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 Cm in the presence of GST-Syn1AC, Gm 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 Gm, Cm 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 Gm 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 Gm 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 [Gm=0.53±0.08 nS (n=5))]. In the HT29-CL19A cells, fluorescence intensity increased to the same degree upon activation of ICFTR 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). Cm 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.

View larger version (17K): [in a new window]   Fig. 5. Detailed time course of the changes in Gm, Cm and FM1-43 intensity in HT29-CL19A cells. It can be clearly seen that Gm activates before exocytosis occurs (as measured by increases in both Cm and FM1-43 intensity), suggesting that ICFTR activation occurs independently of alterations in membrane turnover.

View larger version (13K): [in a new window]   Fig. 6. Inhibition of rapid endocytosis by -Dyn in the gastrointestinal HT29 cells does not change membrane capacitance or conductance in the absence of activation with cAMP. (A) Membrane capacitance experiments were carried out as in Fig. 3. Membrane capacitance and conductance is shown for four representative cells. Transition to the whole-cell configuration is marked by the arrow for each representative capacitance trace. (B) A summary of the capacitance and conductance increases for each of the conditions indicated below each bar. Significant increases over control conditions are indicated above each bar with an asterisk. Data are the average of five to six cells for each condition.

View larger version (24K): [in a new window]   Fig. 7. Disruption of syntaxin 1A and CFTR interactions does not alter the biophysical properties of CFTR; however, open probability increases significantly. (A) Representative single-channel traces of CFTR in 16HBE14o- cells prior to and after addition of 350 nM GST-Syn1AC or 350 nM GST-Syn1AH3 to the cytoplasmic face of inside-out patches. (B) The single-channel conductance for currents in the presence or absence of GST-Syn1AC or GST-Syn1AH3 was 6 pS. The vertical bars are s.e.m. (n ranged from 1 to 8 at each measured potential). (C) Mean open time (o) and mean closed times (c) prior to and after disruption of syntaxin-1A-CFTR interaction by Syn1AC. o (250 mseconds) was unaffected whereas c decreased from 1.95 seconds to 480 mseconds. (D) Open probability (Po) in the absence and presence of GST-Syn1AC was 0.03±0.03 and 0.17±0.05, respectively (n=5 (P<0.05)). Po remained unchanged from 0.07±0.03 to 0.04±0.02 following the addition of the negative control fusion peptide, GST-Syn1AH3 (n=6).