Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl– currents
Anjaparavanda P. Naren, … , Deborah J. Nelson, Kevin L. Kirk
Anjaparavanda P. Naren, … , Deborah J. Nelson, Kevin L. Kirk
Published February 1, 2000
Citation Information: J Clin Invest. 2000;105(3):377-386. https://doi.org/10.1172/JCI8631.
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Article

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl– currents

Abstract

The CFTR Cl– channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl– currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl– currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II–activated Cl– currents in these cells.

Authors

Anjaparavanda P. Naren, Anke Di, Estelle Cormet-Boyaka, Prosper N. Boyaka, Jerry R. McGhee, Weihong Zhou, Kimio Akagawa, Tomonori Fujiwara, Ulrich Thome, John F. Engelhardt, Deborah J. Nelson, Kevin L. Kirk

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Figure 2

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Syntaxin 1A protein is expressed in epithelial cells to a much lesser ex...
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 (3, 4) served as controls (IP from ∼0.5 mg total protein). Genzyme C-CFTR mAb was used both for immunoprecipitation and for detection by immunoblotting.