Cystic Fibrosis Transmembrane Conductance Regulator: A Molecular Model Defines the Architecture of the Anion Conduction Path and Locates a “Bottleneck” in the Pore

Abstract

We developed molecular models for the CFTR chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TMs 3, 6, 9 and 12; with particular attention to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77% to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics (MD) simulation. Pore shape is that of an asymmetric hour glass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow “bottleneck” linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward–facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl− pseudohalide anions, water and urea.

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…On the other hand, applying bulky MTS reagents from the cytoplasmic side of the channel identified position 341 as the accessibility limit (Bai et al 2010;El Hiani and Linsdell 2010). Thus, it appears that the pore is constructed with two fairly accessible internal and external vestibules with a "bottleneck" that traverses only one helical turn (Norimatsu et al 2012). …”

“…Wang and Linsdell 2012). As more experimental and structural data emerge (Norimatsu et al 2012), which TMs contribute to the CFTR pore will become clearer.…”

“…Based on the ATP-bound configuration of ABC exporters, several homology models of CFTR's "open state" were composed Alexander et al 2009;Mornon et al 2009;Norimatsu et al 2012). However, these homology models, like their counterparts of ABC exporters, all lack an uninterrupted pathway for ion permeation.…”

Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

“…The outward facing models 14,[17][18][19][20][21][22][23] were based primarily on the Sav1866 structure as a template, 24 although recently the newly solved ABC transporter structure McjD 25 has been used to model this state. 26 Inward facing models were based either on the bacterial MsbA 27 or on the P-gp 28 structures.…”

The Therapeutic Potential of Small-molecule Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…Building on these studies, Norimatsu et al 57 used cysteine scanning mutagenesis to test the predicted orientation of amino acid side-chains in M3, M6, M9 and M12 from a Sav1866-based molecular model of the CFTR pore. The authors' data suggest that the constriction in the CFTR pore is located towards the extracellular end of the channel with the boundary between the outer vestibule and the constriction located near T338 (M6) and I1131 (M12) and that between the constriction and the inner vestibule located near S341 (M6) and T1134 (M12).…”