期刊
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
卷 304, 期 9, 页码 L582-L592出版社
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajplung.00314.2012
关键词
patch clamp; ubiquitin; secretory pH; Xenopus oocytes; 16HBE14o-
资金
- National Heart, Lung, and Blood Institute [HL031197]
- National Institute of Environmental Health Sciences [5U01ES015676]
- National Institute of Diabetes and Digestive and Kidney Diseases [DK60065]
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R37HL031197, R01HL031197] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK060065] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [U01ES015676] Funding Source: NIH RePORTER
The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl-) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H+) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H+, did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.
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