ABCMdb

PMID: 16484308

Cui L, Aleksandrov L, Hou YX, Gentzsch M, Chen JH, Riordan JR, Aleksandrov AA
The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating.
J Physiol. 2006 Apr 15;572(Pt 2):347-58. Epub 2006 Feb 16., 2006-04-15 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Glu1371Ser Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). Login to comment 6 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Login to comment 7 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Login to comment 8 ABCC7 p.Glu1371Ser Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. Login to comment 126 ABCC7 p.Phe508Cys Influence of F508C substitution on channel gating With a functional cysless CFTR, it became possible to focus on a single cysteine introduced at position 508 which is known to be permissive for maturation of the protein (Du et al. 2005). Login to comment 127 ABCC7 p.Phe508Cys However, as a prelude to this, we first examined the single-channel properties of a F508C Figure 4. Login to comment 132 ABCC7 p.Phe508Cys With F508C in the cysless background there were even larger decreases in Po and increases in τc, with τo remaining unaltered (Fig. 5, second trace). Login to comment 134 ABCC7 p.Phe508Cys This was indicated for cysless F508C in the lower two traces of Fig. 5 using the alternative ligands, dATP and 8BrATP, which resulted in the same increases in τo values relative to that with ATP as a ligand. Login to comment 135 ABCC7 p.Phe508Cys Overall, the data in Fig. 5 revealed that the F508C substitution decelerated the gating of both wild-type and cysless CFTR primarily by prolonging the mean closed Table 1. Login to comment 136 ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys Basic parameters of wild-type and mutant CFTR channels Type Ligand Po τo (ms) τc (ms) γ (pS) n Wild-type CFTR ATP 0.49 ± 0.03 220 ± 10 230 ± 10 12.3 ± 0.2 8 dATP 0.82 ± 0.03 420 ± 10 80 ± 10 12.3 ± 0.2 6 8BrATP 0.75 ± 0.02 510 ± 10 150 ± 10 12.3 ± 0.2 6 8N3ATP 0.58 ± 0.03 710 ± 10 560 ± 10 12.4 ± 0.2 5 F508C ATP 0.23 ± 0.03 230 ± 15 810 ± 10 12.3 ± 0.2 4 Cysless CFTR ATP 0.12 ± 0.03 220 ± 15 1800 ± 120 13.5 ± 0.2 7 dATP 0.21 ± 0.03 410 ± 15 1600 ± 100 13.4 ± 0.2 4 8BrATP 0.24 ± 0.02 520 ± 15 1680 ± 120 13.2 ± 0.2 5 8N3ATP 0.26 ± 0.03 715 ± 15 2100 ± 160 13.4 ± 0.2 3 Cysless F508C ATP 0.04 ± 0.02 210 ± 20 5000 ± 850 13.4 ± 0.2 5 dATP 0.07 ± 0.02 415 ± 20 5200 ± 720 13.5 ± 0.2 3 8BrATP 0.09 ± 0.03 520 ± 20 5600 ± 900 13.5 ± 0.2 3 CFTR, cystic fibrosis transmembrane conductance regulator. Login to comment 139 ABCC7 p.Phe508Cys For cysless F508C, the effective values of τc were estimated as τo(1 - Po)/Po. time. Login to comment 140 ABCC7 p.Phe508Cys While informative, this large prolongation in cysless F508C limits detailed analysis of its gating kinetics. Login to comment 141 ABCC7 p.Glu1371Ser However, it was possible to circumvent this limitation by employing a variant in which the glutamate residue adjacent to the Walker B aspartate in NBD2 was mutated (E1371S). Login to comment 142 ABCC7 p.Glu1371Ser The effect of E1371S substitution on the wild-type CFTR ion channel function is shown in Fig. 6 (upper panel, Po = 0.97 ± 0.02, n = 5) and is in a good agreement with published data (Vergani et al. 2005). Login to comment 144 ABCC7 p.Glu1371Ser While cysless E1371S was not completely locked open, its open probability was increased approximately sevenfold relative to the cysless wild-type protein (compare Fig. 4 upper panel, and Fig. 6 middle panel). Login to comment 145 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser The fact that the essentially non-hydrolytic cysless E1371S channel was able to open and close in a robust mannerisofinterestmechanistically.However,ofpractical importance for the utility of the cysless protein to study the role of the Phe508 residue in gating was the fact that the E1371S substitution also increased the activity of cysless F508C (Fig. 6, lower panel, Po = 0.25 ± 0.03, n = 4). Login to comment 146 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Moreover, it was possible to differentiate better between cysless E1371S and cysless E1371S/F508C by using 8BrATP instead of ATP as a ligand (Fig. 7, first panel, Po = 0.97 ± 0.02, n = 4 and second panel (Fig. 7, second panel, Po = 0.71 ± 0.03, n = 4). Login to comment 150 ABCC7 p.Glu1371Ser To test this assumption we used the cysless E1371S construct that is locked open with 8BrATP as a reference point (Fig. 7, upper panel). Login to comment 152 ABCC7 p.Glu1371Ser The return of an aromatic ring to position 508 by the chemical modification with 50 μm MTSBn restored the locked open state typical of cysless E1371S (Fig. 7,thirdpanel, Po = 0.97 ± 0.02,n = 3).Thisstrongly supports the notion that the aromatic side chain of Phe508 plays a role in channel gating. Login to comment 154 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser This was found to be the case as the positively charged MTSET not only did not lock the cysless E1371S/F508C channel open but completely ablated gating (Fig. 7, lower trace, Po < 0.01, n = 3). Login to comment 163 ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys Influence of F508C on CFTR channel gating Typical records of F508C and cysless F508C single-channel activity driven by different ligands at 30◦C are shown in the middle of each panel. Login to comment 165 ABCC7 p.Phe508Cys Mean closed time for cysless F508C driven by different ligands was roughly estimated as τc = τo(1 - P o)/P o. Login to comment 180 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Improvement of the cysless CFTR ion channel activity by introducing E1371S in NBD2 The upper panel shows typical single-channel activity of the E1371S mutant at 30◦C. Login to comment 181 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser The cysless E1371S single-channel record is shown in the middle panel. The influence of the further introduction of F508C in the cysless E1371S background on the ion channel gating is shown in the lower panel. The mean values of Po ± S.E.M. and number of experiments are shown in the text. All records were done at 30◦C and 2 mM ATP. Login to comment 183 ABCC7 p.Phe508Cys That is why our experiments with the F508C mutation and its chemical modification were done on this particular background. Login to comment 196 ABCC7 p.Glu1371Ser Aromatic side chain at position 508 is required to lock the channel open Cysless E1371S ion channel gated by 2 mM 8BrATP is shown in the upper panel. Login to comment 197 ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser Effect of the introduction of F508C in the cysless E1371S background on the ion channel gating is shown in the second panel. The result of the chemical modification of the cysless E1371S/F508C channel by MTSBn at the cis side on the ion channel gating is shown by the arrow in the third panel. The effect of positively charged 2-trimethylammonioethylmethanethiosulphonate (MTSET) on the cysless E1371S/F508C ion channel function is shown by the arrow in the lower panel. The values of Po before and after chemical modification are shown above the traces. Login to comment 200 ABCC7 p.Phe508Cys Although the F508C variant appeared to mature similarly as wild-type and mediated iodide efflux (Du et al. 2005), we show that at the single-channel level it greatly increased the channelmeanclosedtimeinboththewild-typeandcysless background, suggesting that the phenylalanine side chain plays a role in channel gating. Login to comment 201 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser This could be confirmed using a site II ATPase-inhibited mutant (E1371S) which is locked open in both the wild-type and cysless backgrounds, while the F508C version of cysless E1371S was unable to maintain the locked open state. Login to comment 202 ABCC7 p.Phe508Cys Neither total cysteine removal, nor F508C substitution, affects open state. Login to comment 207 ABCC7 p.Phe508Cys ABCC7 p.Glu1371Ser The ability of F508C version of cysless E1371S to maintain the locked open state was fully restored on modificaton of Cys508 with MTSBn. Login to comment 212 ABCC7 p.Phe508Cys Thus, just as removal of all endogenous cysteines apparently alters the gating response to ATP binding rather than binding itself, this also appears to be the case for the further increment in mean closed time caused by F508C. Login to comment