ABCMdb

PMID: 26496611

Sorum B, Czege D, Csanady L
Timing of CFTR Pore Opening and Structure of Its Transition State.
Cell. 2015 Oct 22;163(3):724-33. doi: 10.1016/j.cell.2015.09.052. Epub 2015 Oct 22., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Asp1370Asn Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. Login to comment 37 ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn To make CFTR gate at equilibrium, we introduced the NBD2 Walker-B mutation D1370N (Figure 1A, bottom, red star) because, among several hydrolysis-disrupting mutations tested, D1370N only slightly reduces the apparent affinity for ATP, and does not prolong open bursts to an extent incompatible with single-channel gating analysis (Csana &#b4; dy et al., 2010). Login to comment 40 ABCC7 p.Asp1370Asn Choice of a Suitable Background Construct for REFER Analysis (A) Domain organizations of WT (top) and cut-DR(D1370N) (bottom) CFTR: TMDs (gray), intracellular loops containing coupling helices (light violet), NBD1 (green), NBD2 (blue), R domain (magenta), membrane (yellow). Login to comment 42 ABCC7 p.Asp1370Asn The D1370N mutation in NBD2 is depicted by a red star. Login to comment 48 ABCC7 p.Asp1370Asn The D1370N mutation abrogates ATP hydrolysis (red cross) and confines gating in saturating ATP to a simple C1 4 O1 equilibrium (red box). Login to comment 53 ABCC7 p.Asp1370Asn Thus, we chose cut-DR(D1370N) as the background construct for our REFER study (Figure 1A, bottom). Login to comment 54 ABCC7 p.Asp1370Asn Gating of cut-DR(D1370N) indeed proved PKA-independent but remained strictly ATP-dependent with an apparent affinity for ATP of 288 &#b1; 27 mM (Figures S1A and S1B). Login to comment 55 ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn Just as for WT (Winter et al., 1994; Zeltwanger et al., 1999; Csana &#b4; dy et al., 2000; Vergani et al., 2003), cut-DR (Csana &#b4; dy et al., 2000; Bompadre et al., 2005), and D1370N (Vergani et al., 2003) CFTR channels, mean open burst duration (tb) of cut-DR(D1370N) proved largely Figure 2. Login to comment 56 ABCC7 p.Asp1370Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Cys ABCC7 p.Thr1246Ala ABCC7 p.Thr1246Val ABCC7 p.Thr1246Pro Timing of Motion at Position 1246 of the NBD1-NBD2 Interface (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations T1246V, T1246P, T1246C, T1246N, and T1246A, respectively, in the same background. Login to comment 63 ABCC7 p.Asp1370Asn ABCC7 p.Tyr275Ser ABCC7 p.Tyr275Phe ABCC7 p.Tyr275Glu ABCC7 p.Tyr275Leu ABCC7 p.Tyr275Lys Timing of Motion at Position 275 of the NBD2-TMD Interface (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations Y275F, Y275E, Y275K, Y275L, and Y275S, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level. Login to comment 74 ABCC7 p.Met348Lys ABCC7 p.Asp1370Asn ABCC7 p.Met348Cys ABCC7 p.Met348Ala ABCC7 p.Met348Asn ABCC7 p.Met348Ile Timing of Motion at Position 348 in the Pore Region (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations M348I, M348K, M348C, M348N, and M348A, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level. Login to comment 79 ABCC7 p.Asp1370Asn Timing of Motion in the Narrow Region of the Pore Studied by Anion Replacement (A) Pairs of segments of inward single-channel current from three patches containing single cut-DR(D1370N) CFTR channels. Login to comment 96 ABCC7 p.Tyr275Leu Kinetic analysis revealed a clear tendency for opposing effects on channel closing and opening rates, both contributing about equally to changes in Po: lengthened tb was mostly associated with shortened tib and (in Y275L) shortened tb with lengthened tib (Figures 3B and 3C). Login to comment 102 ABCC7 p.Met348Lys Interestingly, the M348K mutation only marginally affected gating (Figures 4A-4D) but increased the affinity for pore block by ATP, as reported by pronounced flickery block of single-channel currents in 10 mM ATP (Figure 4A), a bell-shaped ATP dose-dependence of macroscopic currents (Figure S1C, second blue bar), and a current overshoot upon ATP removal from macroscopic patches reflecting rapid unblock (Figure S2F). Login to comment 103 ABCC7 p.Met348Lys ABCC7 p.Met348Lys Of note, even for M348K, the macroscopic current relaxation time constant following ATP removal (i.e., tb in zero ATP; Figure S2F) remained comparable to steady-state tb: thus, even pronounced flickery block of M348K by 10 mM ATP does not delay pore closure, consistent with earlier demonstration that the gate, located on the extracellular side, can readily close while large organic anion blockers remain bound in the intracellular vestibule (Csana &#b4; dy and To &#a8; ro &#a8; csik, 2014). Login to comment 104 ABCC7 p.Met348Glu ABCC7 p.Met348Glu We also replaced the methionine with glutamate, but this M348E mutant could not be studied at a single-channel level due to the presence of subconductance states; however, the rate of macroscopic current relaxation upon ATP removal attested to an acceleration of M348E closing rate comparable to that of the I, C, N, and A mutants (Figures S2G and S2H). Login to comment 108 ABCC7 p.Asp1370Asn Thus, replacement of chloride with other permeant anions might be viewed as a structural perturbation of the ''selectivity filter.`` We therefore studied changes in the pattern of single-channel gating of our background construct cut-DR(D1370N) in response to sudden replacement of cytosolic chloride with nitrate, bromide, or formate. Login to comment 130 ABCC7 p.Asp1370Asn The point representing the cut-DR(D1370N) background construct in chloride is highlighted by a black circle. Login to comment 153 ABCC7 p.Asp1370Asn To study the pore opening step, we therefore employed the D1370N background mutation that truncates the gating cycle to an equilibrium scheme (Figure 1B, red frame). Login to comment 162 ABCC7 p.Asp1370Asn Because gating of cut-DR(D1370N), like that of WT CFTR, is strictly ATP-dependent (Figures S1 and S2), our conclusions do not necessarily apply to the mechanism of the extremely infrequent spontaneous openings observable in the absence of ATP that are promoted by certain mutations (Wang et al., 2010) and drugs (Jih and Hwang, 2013). Login to comment 165 ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn EXPERIMENTAL PROCEDURES pGEMHE-CFTR(837-1480(D1370N)) was constructed from pGEMHE-CFTR(837-1480), mutations at positions 275 and 348 were introduced into pGEMHE-CFTR(1-633) (Csana &#b4;dy et al., 2000), and mutations at position 1246 into pGEMHE-CFTR(837-1480(D1370N)) using Stratagene QuickChange. Login to comment