Abstract
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.
Highlights
Two salt bridges, Arg347–Asp924 and Arg352–Asp993, have been identified in CFTR, but the timing of their interaction remains unknown
Arg347 Forms a Salt Bridge with Asp924 but Does Not Stabilize the Full Open State— Cotten and Welsh first reported that arginine 347 of TM6 forms a salt bridge with aspartic acid 924 of TM8, their results suggested that the double mutation R347D/D924R rescued the channel to a stable open state that exhibits a smaller single channel amplitude, which is reminiscent of the s2 open state of wild type CFTR (WT-CFTR) [14]
1) Large conductance calciumactivated Kϩ channels exhibit subconductance states, and flickers in the full open state result from channel pore conformational changes; 2) short lived subconductance states in Kv2.1 channels were found to be due to subunit interactions, and these processes are allosterically coupled; 3) subconductance states in L-type Ca2ϩ channels are produced from the different permeant ions, including Ca2ϩ, Ba2ϩ, and Liϩ, that differ in binding affinity in the pore; 4) subconductance channel behavior in Shaker K channels, cyclic nucleotide-gated channels, and glutamate receptor channels has been associated with channel activation and inactivation (30 –34)
Summary
Arg347–Asp924 and Arg352–Asp993, have been identified in CFTR, but the timing of their interaction remains unknown. The Arg352-Asp993 salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. CFTR channel pore opening is accomplished by R domain phosphorylation and the binding of ATP at the NBDs, where the two MSDs are driven to change conformation to allow anions to flow It remains unclear how the CFTR channel pore moves during the gating cycle, including whether it passes through multiple conductance states or switches directly between fully “closed” (c) and fully “open” (f) states. Our results demonstrate that Arg347, Asp924, and Asp993 form a triangular salt bridge early in channel openings, whereas Arg352 and Asp993 interact late in channel opening These results contribute to our understanding of how CFTR gained ion channel function despite its origin in the ABC transporter superfamily
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