Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium

Abstract

Chloride (Cl-) secretion by the airway epithelium regulates, in part, the quantity and composition of the respiratory tract fluid, thereby facilitating mucociliary clearance. The rate of Cl- secretion is controlled by apical membrane Cl- channels. Apical Cl- channels are opened and Cl- secretion is stimulated by a variety of hormones and neurotransmitters that increase intracellular levels of cyclic AMP (cAMP). In cystic fibrosis (CF), a common lethal genetic disease of Caucasians, airway, sweat-gland duct, secretory-coil and possibly other epithelia are anion impermeable. This abnormality may explain several of the clinical manifestations of the disease. The Cl- impermeability in CF-airway epithelia has been localized to the apical cell membrane, where regulation of Cl- channels is abnormal: hormonal secretagogues stimulate cAMP accumulation appropriately but Cl- channels fail to open. Here we report that the purified catalytic subunit of cAMP-dependent protein kinase plus ATP opens Cl- channels in excised, cell-free patches of membrane from normal cells, but fails to open Cl- channels in CF cells. These results indicate that in normal cells, the cAMP-dependent protein kinase phosphorylates the Cl- channel or an associated regulatory protein, causing the channel to open. The failure of CF Cl- channels to open suggests a defect either in the channel or in such an associated regulatory protein.

Anoctamin 6 is an essential component of the outwardly rectifying chloride channel

“…contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: karl.kunzelmann@vkl.uniregensburg.de.…”

“…These channels are activated upon excision of the cell membrane from the intact cell and after strong depolarization (1)(2)(3)(4)(5). It is therefore unclear whether ORCC contributes to epithelial salt transport and whether it has a role in cystic fibrosis (1)(2)(3)(4)6). Subsequent reports claimed that the cystic fibrosis transmembrane conductance regulator (CFTR) and ORCC are distinct proteins with a regulatory relationship and that both channels contribute to the cAMP-dependent Cl − conductance (5,7,8).…”

“…These channels are activated upon excision of the cell membrane from the intact cell and after strong depolarization (1)(2)(3)(4)(5). It is therefore unclear whether ORCC contributes to epithelial salt transport and whether it has a role in cystic fibrosis (1)(2)(3)(4)6).…”

“…O utwardly rectifying chloride (Cl − ) channels (ORCC, ICOR, ORDIC) of intermediate single-channel conductance of about 50 pS are expressed abundantly (1)(2)(3). These channels are activated upon excision of the cell membrane from the intact cell and after strong depolarization (1)(2)(3)(4)(5).…”

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

“…In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion permeability with the electrophysiological signature of CFTR [13] [14,15].…”