Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

The Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cystic Fibrosis Transmembrane Conductance Regulator Inhibition by Cigarette Smoke

“…In addition to biochemical changes in CFTR expression that occur on a chronic time frame, our results point out the importance of acute inhibition of CFTR activity through altered channel gating by CSE, and that its underlying molecular mechanism may be due to covalent modifications of the CFTR protein itself. Acute inhibition of I SC was immediate and robust, even in native epithelia, supporting previous observations of altered CFTR gating observed in CFTR-transduced Xenopus oocytes (22). Reversal by ivacaftor in the same studies lends credence to this observation, and rule out loss of channels as the primary cause (in addition to the nature of excised patch studies, where protein recycling machinery is absent).…”

“…Acute blockade of CFTR-dependent currents that were not dependent on CFTR expression levels suggested that acute effects on CFTR channel gating may be occurring, as also suggested in a report by Moran and colleagues (22). To confirm, we conducted whole-cell and membrane patch clamp studies on HEK293 cells expressing wildtype CFTR.…”

Acquired Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction in Chronic Bronchitis and Other Diseases of Mucus Clearance

“…6,7,59 Single channel conductance studies demonstrated the deleterious effect of CSE on CFTR open channel probability (Po). 60 CSE exposure also reduced mature, fully-glycosylated (the post ER glycoform) CFTR C-band by Western blot, and was accompanied by reductions in CFTR mRNA expression by Real-Time RT-PCR. 6 These findings are concordant with results seen in Calu-3 pulmonary glandular monolayers with high-level wild type CFTR expression.…”

Airway hydration and COPD

“…In single channel patch clamp recordings, acrolein has been demonstrated to directly affect CFTR gating [172]. Cigarette smoke is acidic, and 50 standard cigarettes drawn through 50 mls of Frog Ringer solution acidified the solution by about 2 pH units [173]. This cigarette-exposed acidic media inhibited the activity of CFTR expressed in Xenopus laevis oocytes [125].…”