ABCMdb

PMID: 15729345

Vergani P, Lockless SW, Nairn AC, Gadsby DC
CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains.
Nature. 2005 Feb 24;433(7028):876-80., 2005-02-24 [PubMed]

Sentences

No. Mutations Sentence Comment

71 ABCC7 p.Glu1371Gln Time constants for current decay fit lines (blue): WT, t ¼ 0.45 s; E1371Q, t ¼ 476 s. Note the fivefold expanded timescale for the WT record. Login to comment 73 ABCC7 p.Glu1371Gln Evidence supporting our speculation (Fig. 1a) that the CFTR-channel open state corresponds to the dimerized NBD conformation is provided by the approximately 1,000-fold stabilization of the open-burst state that results from mutation of the possible catalytic base4,17 , Glu 1371 (a glutamate in the NBD2-head 'Walker B` motif), to Gln (E1371Q; Fig. 1b). Login to comment 74 ABCC7 p.Glu1371Gln Because CFTR channels do not open without ATP (see below), current decay upon the removal of ATP reflects channel closing, and its time course measures the open burst duration; closing was complete within about 1 s of ATP withdrawal for wild-type (WT) CFTR channels (mean open burst duration was less than 0.5 s), but had a time constant of 411 ^ 64 s (mean ^ s.e.m., n ¼ 16) for mutant E1371Q channels (Fig. 1b). Login to comment 85 ABCC7 p.Arg555Lys ABCC7 p.Arg555Gln c, Mutations at Arg 555 in NBD1 'tail` affect mean open burst (R555Q) and closed interburst (R555K) dwell times. Login to comment 86 ABCC7 p.Arg555Gln ABCC7 p.Arg555Gln R555Q opened with rates comparable to WT (R555Q, tib ¼ 2.84 ^ 0.53 s (15)) despite expected loss of the interfacial hydrogen bond, suggesting possible compensation by other factors, for example the removal of repulsive electrostatic forces. Login to comment 90 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn b, Representative records from WT, single mutants R555K and T1246N, and double mutant R555K T1246N. Login to comment 95 ABCC7 p.Arg555Gln ABCC7 p.Arg555Gln We first mutated Arg 555, and found that the charge-removing mutation R555Q slowed channel closing (mean open burst duration was 3.20 ^ 0.35 s (n ¼ 18) for R555Q, compared with 0.43 ^ 0.02 s (n ¼ 32) for WT; Fig. 2c), consistent with Arg 555 being part of the composite NBD2 catalytic site, where the ATP hydrolysis that times closing of WT channels occurs. Login to comment 97 ABCC7 p.Arg555Lys Accordingly, the charge-conserving mutation R555K did not affect open burst duration (mean 0.39 ^ 0.04 s (n ¼ 26)). Login to comment 98 ABCC7 p.Arg555Lys However, it substantially prolonged closed interburst duration (inversely related to opening rate) from 2.29 ^ 0.46 s (n ¼ 16) for WT to 8.53 ^ 1.23 s (n ¼ 15) for R555K (Fig. 2c, all measured at saturating [ATP]). Login to comment 99 ABCC7 p.Arg555Lys On the basis of the evolutionary evidence suggesting the involvement of the side chain at this position in a conserved interaction (Fig. 2b), this slowing of channel opening could be explained if the R555K mutation were to weaken or remove a hydrogen bond between NBD1 and NBD2 that is absent in the closed, ground, state but present in the transition state for the channel opening reaction; the resulting destabilization of the transition state would increase the activation free energy (DG‡ ) for channel opening and hence decrease the opening rate. Login to comment 100 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Arg555Thr To quantify this suspected interaction between Arg 555 and Thr 1246 side chains (Fig. 2a), we applied double mutant-cycle analysis6,21 (see Supplementary Information), after mutating Arg 555 to Lys and Thr 1246 to Asn, both individually and jointly. Login to comment 102 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn If the two residues do not interact, the effects of mutating Arg 555 to Lys should be the same in a Thr 1246 background as in a T1246N background (and vice versa); that is, the effects of the single mutations should be independent and hence additive, and mutation-linked changes on parallel sides of the cycles should thus be equal. Login to comment 112 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Lys1250Arg Current levels of the triple mutant R555K T1246N K1250R did not change when [ATP] was increased to 10 mM, indicating that 5 mM [ATP] was saturating. Login to comment 117 ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn The apparent affinity for ATP was little influenced by the mutation R555K (R555K K0.5 ¼ 71 ^ 14 mM versus WT K0.5 ¼ 55 ^ 5 mM), but was reduced by the mutation T1246N (T1246N K0.5 ¼ 261 ^ 49 mM) by the same extent in the WT background as in the R555K background (R555K T1246N K0.5 ¼ 257 ^ 51 mM). Login to comment 124 ABCC7 p.Arg555Lys The slowing of opening caused by the R555K mutation (Fig. 2c, above) corresponded to a 1.4 ^ 0.4kT increase in the activation energy barrier. Login to comment 125 ABCC7 p.Thr1246Asn The T1246N mutation also greatly slowed channel opening, increasing the energy barrier by 2.5 ^ 0.4kT. Login to comment 132 ABCC7 p.Lys1250Arg In other ABC-ATPases, mutating the key lysine in the phosphate-binding loop to arginine drastically reduces or abolishes hydrolysis (see, for example, ref. 24) and, as would be predicted if hydrolysis at CFTR`s NBD2 catalytic site were markedly slowed, CFTR channels carrying the corresponding mutation (K1250R) have prolonged open burst durations (Fig. 4e; mean time constant of current decay upon ATP removal t ¼ 9.3 ^ 0.5 s; n ¼ 49). Login to comment 133 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Lys1250Arg ABCC7 p.Lys1250Arg Introducing the T1246N mutation into the K1250R background decreased Po, corresponding to destabilization of the open burst state by 2.5 ^ 1.0kT with respect to the closed state. However, adding the R555K mutation to T1246N-K1250R channels restored high stability of the open state (Fig. 4e, f). Login to comment 157 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn For constructs with very low Po (R555K and T1246N), we could not exclude the presence of unseen channels in the patch (even though the records lasted on average 6-7 min). Login to comment 158 ABCC7 p.Arg555Lys ABCC7 p.Thr1246Asn The prolonged tib values we extract for R555K and T1246N channels are therefore most probably underestimates, and the real effects of the mutations are more severe (and, hence, jDDG‡ int(opening)j is actually larger) than the values we report. Login to comment