PMC

Syntaxin 1A does not influence Cl^– currents in MTE cells induced by CaMK II. Cl^– currents were activated by inclusion of 50 ng CaMK II in the pipette in the presence or absence of 350 nM GST-syn 1AΔC (refer to bars). In some cells, currents were activated with cAMP cocktail with or without CAMK II and GST-syn 1AΔC. Numbers of experiments are indicated in parentheses. Holding potential: +110 mV.

Pharmacology and I–V behavior of Cl^– currents in tracheal epithelial cells potentiated by GST-syn 1AΔC. (a) Basal Cl^– currents in presence of GST-syn 1AΔC (350 nM). (b) Large Cl^– currents in the presence of GST-syn 1AΔC and cAMP cocktail. (c) No effect of DIDS (1 mM) on Cl^– currents in the presence of GST-syn 1AΔC. (d) Complete block of GST-syn 1AΔC-potentiated Cl^– currents by DPC (1 mM). (e) Current-voltage relationships for a–d. All data are for the same cell.

Immunofluorescence localization of syntaxin 1A (a) and 3 (c) in human bronchus. Nonimmune control lacking primary antibody (e) and inhibition of staining by fusion peptide competition (b and d) are also shown. Arrows represent the apical pole of the bronchial epithelium. Affinity-purified syntaxin 1A and 3 polyclonal antibodies were used at 5 μg/mL. Transmitted light micrographs were generated using Nomarski optics.

Mouse CFTR Cl^– channels physically and functionally interact with syntaxin 1A in an isoform-specific manner. (a) CFTR Cl^– current activity in MTE cells is potentiated by GST-syn 1AΔC but not by other syntaxin fusion proteins (350 nM). Inset shows the binding of mouse CFTR to mouse syn 1AΔC in a pulldown assay performed as described elsewhere (, ). (b) GST-syn 1AΔC (350 nM) does not potentiate CFTR Cl^– current activity in L cells stably expressing recombinant CFTR (black bars). Transient expression of membrane-anchored syntaxin 1A reduces CFTR Cl^– current, which can be reversed by GST-syn 1AΔC fusion protein but not GST alone (750 nM). Numbers of experiments are indicated in parentheses. Holding potential: + 110 mV.

GST-syn 1AΔC and GST-Munc-18 potentiate cAMP-dependent Cl^– currents in MTE cells and human nasal polyps. (a) GST-syn 1AΔC (350 nM), GST-Munc-18a (200 nM), and GST alone (750 nM) were included in the patch pipette in the absence or presence of a cAMP-cocktail (50 μM forskolin, 50 μM IBMX, 100 μM cpt-cAMP). The cAMP cocktail was added to the bath 5–10 minutes after seal formation. In some cases, the patch pipette also contained a CFTR neutralizing antibody (). GST-syn 1AΔH3 (350 nM), which lacks H3 domain of syntaxin 1A (amino acids 194–266) required for CFTR binding (), had no effect on Cl^– currents. (b) GST-syn 1AΔC (350 nM) also potentiates cAMP-mediated Cl^– currents in human nasal polyps. Numbers of experiments are indicated in parentheses. Error bars are SEMs. Holding potential: +110 mV. Currents (pA) are normalized to cell capacitance (pF).

Syntaxin 1A is expressed in epithelial cells that normally express CFTR. (a) Isoform specificity of syntaxin 1A and syntaxin 3 antibodies (each lane: 50 ng fusion protein cleaved free of GST by thrombin; ref. ). (b) Expression of syntaxin 1A and syntaxin 3 proteins in MTE cells and HBE cells. The original isolate (P0) and primary culture (P1) of HBE cells were both analyzed for syntaxin protein expression. HT29-CL19A colonic carcinoma cells and mouse L fibroblasts were also analyzed for syntaxin protein expression. Antibodies for a and b were as follows: 14D8 syntaxin 1A mAb (0.1 μg/mL) and affinity-purified syntaxin 3 polyclonal (0.1 μg/mL). (c) Expression of CFTR protein, syntaxin 1A protein, and syntaxin 3 protein in freshly isolated MIE cells. MIE cells were isolated and fractionated as described in the methods. CFTR protein was detected by immunoprecipitation from cell lysate (200 μg protein) as described in Methods. Syntaxin 1A and 3 were detected by immunoblotting cell lysates (50 μg protein each) using 14D8 monoclonal and syntaxin 3 affinity-purified polyclonal antibodies. (d) Profile of alkaline phosphatase activity (villus marker) in corresponding fractions.

Syntaxin 1A protein is expressed in epithelial cells to a much lesser extent than in brain, but at large mole excess over CFTR. (a) Comparison of syntaxin 1A protein expression in rat brain, HT 29-CL19A cells, native MIE cells, and native MTE cells. Monoclonal syntaxin 1A antibody (14D8) was used at 0.1 μg/mL. MIE cells were pooled across Percoll gradient fractions. (b) Quantitation of syntaxin 1A protein in rat brain lysate (14D8 mAb) determined from a standard curve generated using the indicated amounts of syn 1AΔC protein cleaved free of GST. (c) Quantitation of syntaxin 1A in HT29-CL19A cells. Syn1AΔC was used as a standard and 14D8 mAb for detection. (d) Quantitation of CFTR protein in HT29-CL19A cells. MBP-C-CFTR was used as standard (see Methods). Genzyme C-CFTR monoclonal was used at 0.25 μg/mL dilution. (e) Quantitation of syntaxin 3 protein in HT29-CL19A cells. Syn 3ΔC (cleaved to remove GST) was used as standard. Affinity-purified syntaxin 3 polyclonal antibody was used for blotting (0.1 μg/mL). (f) Quantitation of Munc-18 protein in HT29-CL19A cells. GST-Munc-18 was used as standard, and affinity-purified Munc-18 monoclonal antibody was used for blotting at 0.25 μg/mL. (g) Relative amounts of CFTR protein in MIE cells (IP from 1,000 μg lysate), MTE cells (IP from 2,000 μg lysate), and HT29-CL19A cells (IP from 1,000 μg lysate). Cos-7 cells transiently expressing or not expressing recombinant CFTR (, ) served as controls (IP from ∼0.5 mg total protein). Genzyme C-CFTR mAb was used both for immunoprecipitation and for detection by immunoblotting.