Probing the CFTR Chloride Channel with Channel Permeant Thio-Reactive Reagent Au(CN)2-

Abstract

Previous cysteine scanning experiments on the CFTR channel have identified transmembrane segments (TM) 1, 3, 6, 9, and 12 as pore-lining components. using bulky MTS reagents, we and others have demonstrated a restrictive region in TM 1 and 6 that prevents MTS from passing through the CFTR pore. State-dependent modification data also suggest a lack of a physical gate cytoplasmic to this restrictive region. Here we further tested gated access of engineered cysteines in TM6 by using channel permeant probes, like [Ag(CN)2]- and [Au(CN)2]-. We first focused our effort on two positions, 338 and 344, located external and internal respectively to the restrictive region. In excised inside-out patches, application of [Au(CN)2]- causes a biphasic decay of the currents for both I344C- and T338C-CFTR. Since the fast phase is reversible and also seen with the cysless background, it is likely the slow phase reflects covalent modification of introduced cysteines by [Au(CN)2]- while the fast phase is due to blockade of the pore by the negatively charged reagent. Measurements of the modification rate in the presence or absence of ATP suggest that I344C can be accessed in both the open and the closed states, consistent with our previous studies using channel impermeant probes. However, preliminary data suggest the modification rate for C338 in the presence of ATP is ∼10-fold higher than that in the absence of ATP. More extensive studies are in process to unravel the underlying gating motion of CFTR's TMs.