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The structural composition of CFTR's anion permeation pathway has been proposed to consist of a short narrow region, flanked by two wide inner and outer vestibules, based on systematic cysteine scanning studies using thiol-reactive probes of various sizes. Although these studies identified several of the transmembrane segments (TMs) as pore lining, the exact spatial relationship between pore-lining elements remains under debate. Here, we introduce cysteine pairs in several key pore-lining positions in TM1, 6, and 12 and use Cd(2+) as a probe to gauge the spatial relationship of these residues within the pore. We find that inhibition of single cysteine CFTR mutants, such as 102C in TM1 or 341C in TM6, by intracellular Cd(2+) is readily reversible upon removal of the metal ion. However, the inhibitory effect of Cd(2+) on the double mutant 102C/341C requires the chelating agent dithiothreitol (DTT) for rapid reversal, indicating that 102C and 341C are close enough to the internal edge of the narrow region to coordinate one Cd(2+) ion between them. We observe similar effects of extracellular Cd(2+) on TM1/TM6 cysteine pairs 106C/337C, 107C/337C, and 107C/338C, corroborating the idea that these paired residues are physically close to each other at the external edge of the narrow region. Although these data paint a picture of relatively symmetrical contributions to CFTR's pore by TM1 and TM6, introducing cysteine pairs between TM6 and TM12 (348C/1141C, 348C/1144C, and 348C/1145C) or between TM1 and TM12 (95C/1141C) yields results that contest the long-held principle of twofold pseudo-symmetry in the assembly of ABC transporters' TMs. Collectively, these findings not only advance our current understanding of the architecture of CFTR's pore, but could serve as a guide for refining computational models of CFTR by imposing physical constraints among pore-lining residues.

Spatial positioning of CFTR’s pore-lining residues affirms an asymmetrical contribution of transmembrane segments to the anion permeation pathway