Potentiation of CFTR Gating by an Energetically Additive Mechanism

Abstract

CFTR is the only member of the ATP-binding cassette (ABC) protein superfamily that functions as an ion channel. Once its regulatory (R) domain is phosphorylated, CFTR will harness the free energy from ATP binding/hydrolysis in the nucleotide-binding domains (NBDs) to power the conformational changes of the gate located in the transmembrane domains (TMDs). We inadvertently found that some permeant anions such as nitrate or bromide, when applied to the cytoplasmic side of the channel, can increase the Po of CFTR by raising slightly the opening rate but decreasing considerably the closing rate. Since nitrate also decelerates non-hydrolytic closing, we conclude that the effect of nitrate is independent of ATP hydrolysis. Surprisingly, despite their markedly different physical properties, the gating effects of nitrate are remarkably similar to those of VX-770, a well-characterized CFTR potentiator now used in clinics. Nonetheless, while VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation but via two distinct sites of action. Since nitrate increases the Po of ΔR- as well as ΔNBD2-CFTR, we exclude the R domain and NBD dimer as potential targets for nitrate. Furthermore, for both G551D- and ΔNBD2-CFTR, two mutants with extremely low Po, we show that the fold-increase of the Po by nitrate plus VX-770 is virtually the same as the product of fold increases by individual reagents. Collectively, our data suggest that nitrate and VX-770 independently affect CFTR gating by shifting the equilibrium of gating conformational changes of the TMDs towards the open channel configuration. These results may not only shed light on the future development of novel therapeutical reagents, but also provide insights into the coupling mechanism of gate opening/closing and NBD dimerization/dissociation.