title: Localizing a gate in CFTR.
abstract: Experimental and computational studies have painted a picture of the chloride permeation pathway in cystic fibrosis transmembrane conductance regulator (CFTR) as a short narrow tunnel flanked by wider inner and outer vestibules. Although these studies also identified a number of transmembrane segments (TMs) as pore-lining, the exact location of CFTR's gate(s) remains unknown. Here, using a channel-permeant probe, [Au(CN)2](-), we provide evidence that CFTR bears a gate that coincides with the predicted narrow section of the pore defined as residues 338-341 in TM6. Specifically, cysteines introduced cytoplasmic to the narrow region (i.e., positions 344 in TM6 and 1148 in TM12) can be modified by intracellular [Au(CN)2](-) in both open and closed states, corroborating the conclusion that the internal vestibule does not harbor a gate. However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2](-) in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2](-) when the open probability is markedly reduced by introducing a second mutation, G1349D. As [Au(CN)2](-) and chloride ions share the same permeation pathway, these results imply a gate is situated between amino acid residues 337 and 344 along TM6, encompassing the very segment that may also serve as the selectivity filter for CFTR. The unique position of a gate in the middle of the ion translocation pathway diverges from those seen in ATP-binding cassette (ABC) transporters and thus distinguishes CFTR from other members of the ABC transporter family.
authors: Dalton Cardiovascular Research Center and Departments of Biological Engineering and.
authors: Xiaolong
authors: X
authors: Gao
authors: Dalton Cardiovascular Research Center and Departments of Biological Engineering and Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO 65211 HwangT@health.missouri.edu.
authors: Tzyh-Chang
authors: TC
authors: Hwang
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003485:Cyanates / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D006046:Gold / Q000737:chemistry / Q000378:metabolism; D015640:Ion Channel Gating; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D003485:Cyanates; D050505:Mutant Proteins; D013438:Sulfhydryl Compounds; C043041:gold cyanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006046:Gold; D003545:Cysteine
keywords: ABC transporters; anion channels; cystic fibrosis; gating