ABCMdb

PMID: 25675504

Gao X, Hwang TC
Localizing a gate in CFTR.
Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2461-6. doi: 10.1073/pnas.1420676112. Epub 2015 Feb 9., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gly1349Asp However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2]- in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2]- when the open probability is markedly reduced by introducing a second mutation, G1349D. Login to comment 68 ABCC7 p.Ile344Cys (C) Reaction between I344C-CFTR and 1 mM [Au(CN)2]- in the presence or absence of ATP (see Results for details). Login to comment 70 ABCC7 p.Ile344Cys (D) State-independent reactivity of I344C-CFTR to [Au(CN)2]- . Login to comment 78 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys State-Dependent Reactivity of T338C, F337C, and R334C Implicates the Location of a Gate for CFTR. Login to comment 84 ABCC7 p.Thr338Cys As shown in Fig. 2A, the application of just 50 bc;M [Au(CN)2]- in the presence of ATP abolished over 90% of the T338C-CFTR current in an inside-out patch, and this reduction of the current is attributed to the stable coordination between 338C and the probe because removal of [Au(CN)2]- from the solution failed to recover the current. Login to comment 87 ABCC7 p.Thr338Cys To the contrary, the macroscopic current of T338C-CFTR remained almost constant when [Au(CN)2]- was perfused to the patch in the absence of ATP (Fig. 2B). Login to comment 88 ABCC7 p.Arg334Cys ABCC7 p.Phe337Cys Similar observations were made for F337C-CFTR and R334C-CFTR (Fig. S4 A-D). Login to comment 89 ABCC7 p.Lys335Cys For reasons unclear at this juncture, application of 1 mM [Au(CN)2]- to inside-out patches containing K335C-CFTR only resulted in reversible block, although this residue has been previously implicated as pore-lining (Fig. S4E) (21). Login to comment 90 ABCC7 p.Lys335Cys In addition, 1 mM [Au(CN)2]- applied in the absence of ATP also hardly altered the K335C-CFTR current in inside-out patches (Fig. S4F). Login to comment 96 ABCC7 p.Gly1349Asp ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys Our previous studies demonstrated that a disease-associated mutation G1349D could decrease the Po of CFTR by ~10-fold (34) without affecting trafficking of the channel (34, 35); we thus engineered this mutation into R334C, K335C, F337C, and T338C backgrounds. Login to comment 97 ABCC7 p.Gly1349Asp ABCC7 p.Thr338Cys As shown in Fig. S5, indeed, introducing the G1349D mutation into T338C-CFTR lowered the Po and decreased the reaction rate by ~10-fold, which can be interpreted as a limited accessibility of the side chain of 338C in the closed state. Login to comment 98 ABCC7 p.Gly1349Asp However, the reaction rate of extracellularly applied [Au(CN)2]- for the 337C/G1349D mutant is slightly but noticeably faster than that of the 337C mutant (Fig. S5B), suggesting that the 337C side chain is better exposed in the closed state. Login to comment 99 ABCC7 p.Arg334Cys This slight difference in reaction rates between single and double mutants was also seen for R334C, which is one helical turn external to positions 337. Login to comment 100 ABCC7 p.Arg334Cys As shown in Fig. 3A, in the presence of forskolin, 20 bc;M external [Au(CN)2]- readily abolished whole-cell R334C-CFTR currents. Login to comment 102 ABCC7 p.Gly1349Asp ABCC7 p.Arg334Cys Similar experiments were performed with the double mutant R334C/G1349D (Fig. 3B). Login to comment 103 ABCC7 p.Gly1349Asp ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Fitting the current decays upon addition of [Au(CN)2]- yielded the reaction rates of 403 &#b1; 20 /M/s (n = 7) and 537 &#b1; 56 /M/s (n = 6) for R334C and R334C/G1349D, respectively. Login to comment 104 ABCC7 p.Gly1349Asp ABCC7 p.Arg334Cys Although the Po of R334C-CFTR cannot be assessed due to a greatly reduced single-channel amplitude, by comparing macroscopic current amplitudes in a large number of patches (Fig. 3E), we verified a more than 10-fold decrease of Po by introducing the G1349D mutation. Login to comment 107 ABCC7 p.Lys335Cys When 1 mM [Au(CN)2]- was applied to the external side of the cell expressing K335C-CFTR mutants, the whole-cell current decreased in two steps (Fig. 3C). Login to comment 109 ABCC7 p.Lys335Cys These results, together with the only blocking effect of 1 mM [Au(CN)2]- being seen on K335C-CFTR in inside-out patches in the presence of ATP (Fig. S4E), indicate that the side chain of 335C is not well exposed in the open state. Login to comment 110 ABCC7 p.Gly1349Asp ABCC7 p.Lys335Cys However, after G1349D was introduced into K335C-CFTR to lower its Po, 50 bc;M [Au(CN)2]- could readily react with 335C with a reaction rate of 1,809 &#b1; 201 /M/s (n = 5) (Fig. 3D). Login to comment 114 ABCC7 p.Thr338Cys State-dependent reactivity of T338C-CFTR to internal [Au(CN)2]- . Login to comment 115 ABCC7 p.Thr338Cys (A) In the presence of ATP, internal [Au(CN)2]- irreversibly decreases T338C-CFTR currents. Login to comment 117 ABCC7 p.Thr338Cys (B) Contrary to that seen in A, T338C-CFTR currents were not significantly altered when the same concentration [Au(CN)2]- was applied in the absence of ATP for a total of 36 s. considering the side chain of 344C is accessible by intracellular [Au(CN)2]- regardless of whether the channel is open or closed (Fig. 1 C and D), we propose a gate residing in between positions 337 and 344 along TM6 for CFTR (Fig. 1A). Login to comment 140 ABCC7 p.Arg334Cys (A) External application of 20 bc;M [Au(CN)2]- diminished over 80% of forskolin (Fsk)-activated R334C-CFTR currents. Login to comment 143 ABCC7 p.Gly1349Asp ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys (B) [Au(CN)2]- reacted with R334C/G1349D at a faster rate than that with R334C. Login to comment 146 ABCC7 p.Lys335Cys (C) Reaction of K335C-CFTR channels with [Au(CN)2]- . Login to comment 148 ABCC7 p.Lys335Cys The addition of 1 mM [Au(CN)2]- led to a biphasic decay of forskolin-activated K335C-CFTR currents: a faster phase of blockade and a slower phase of ligand exchange reaction. Login to comment 149 ABCC7 p.Gly1349Asp ABCC7 p.Lys335Cys (D) Reaction of K335C/G1349D-CFTR with [Au(CN)2]- . Login to comment 153 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys (E) Comparisons of the mean current amplitude between R334C-CFTR and R334C/G1349D-CFTR and between K335C-CFTR and K335C/G1349D-CFTR in excised inside-out patches. Login to comment 154 ABCC7 p.Gly1349Asp Note that a ~10-fold difference in the mean current amplitude-hence a ~10-fold change in Po-was seen with the introduction of the G1349D mutation, as shown previously for WT-CFTR (see Results for details). Login to comment 192 ABCC7 p.Gly1349Asp ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Ile344Cys ABCC7 p.Phe337Cys ABCC7 p.Asn1148Cys [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment 313 ABCC7 p.Thr338Cys Serrano JR, et al. (2006) CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore. Biophys J 91(5):1737-1748. Login to comment 318 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Bompadre SG, Sohma Y, Li M, Hwang TC (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment