Conformational changes opening and closing the CFTR chloride channel: Insights from cysteine scanning mutagenesis

Abstract

Cystic fibrosis, the most common lethal genetic disease affecting young people in North America, is caused by failure of the chloride ion channel known as CFTR (cystic fibrosis transmembrane conductance regulator). CFTR belongs to the large family of ATP-binding cassette (ABC) membrane transporters. In CFTR, ATP-driven events at the nucleotide-binding domains (NBDs) open and close a gate that controls chloride permeation. However, the conformational changes concomitant with opening and closing of the CFTR gate are unknown. Diverse techniques including substituted cysteine accessibility method, disulfide cross-linking, and patch-clamp recording have been used to explore CFTR channel structure. Here, we consider the architecture of both the open and the closed CFTR channel. We review how CFTR channel structure changes between the closed and the open channel conformations and portray the relative function of both cytoplasmic and vestigial gates during the gating cycle. Understanding how the CFTR channel gates chloride permeation is central for understanding how CFTR defects lead to CF. Such knowledge opens the door for novel ways to maximize CFTR channel activity in a CF setting.

Molecular structure of the ATP-bound, phosphorylated human CFTR

“…3 A – D ). Consistent with this conformational change, residues L102C, F337C, T338C, and N1138C in cysteine-free constructs are accessible to intracellular cysteine reactive reagents only in the channel-open state (20–22).…”

“…The radius of the opening is about 1.2 Å, too small to permit ion or water permeation. Among the residues surrounding this opening, S341 and T338 have been proposed to gate the pore (20, 21). Three positively charged residues, R104, R334, and K335, cluster on the extracellular side of the opening (Fig.…”

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

“…A transition from a TM(287–288)-like to a McjD-like conformation in CFTR would thus likely predict a decrease in reactivity of a thiolate at position 1148 both toward bulky cytosolic reagents ( 73 ) and toward smaller permeant probes ( 85 ). The need to negotiate relatively tight lateral tunnels on the cytosolic permeation pathway to gain access to sites deep within the inner vestibule might explain the “paradoxical” observation that the inner vestibule appears to become less accessible upon channel opening ( 73 , 112 ).…”

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

“…21) and that includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane (22). This Cys-less variant, which we have used extensively in previous studies of CFTR pore structure and function (8), has functional pore properties very similar to those of wild-type CFTR (23). Additional mutations were introduced into the Cys-less background using the QuikChange site-directed mutagenesis system (Agilent Technologies, Santa Clara, CA) and verified by DNA sequencing.…”