ABCMdb

ABCC7 p.Asp993Arg

ClinVar:	c.2977G>T , p.Asp993Tyr ? , not provided
CF databases:	c.2977G>T , p.Asp993Tyr (CFTR1) D , The above mutation was found by DGGE and then direct sequencing of DNA from a patient with severe phenotype from Southern France. c.2978A>G , p.Asp993Gly (CFTR1) ? , The mutation was detected by DHPLC analysis and characterized by direct sequencing
Predicted by SNAP2:	A: D (71%), C: D (71%), E: D (66%), F: D (80%), G: D (80%), H: D (80%), I: D (75%), K: D (91%), L: D (85%), M: D (75%), N: D (80%), P: D (91%), Q: D (80%), R: D (91%), S: D (71%), T: D (80%), V: D (80%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, E: N, F: D, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D,

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[hide] Cystic fibrosis-associated mutations at arginine 3... J Biol Chem. 1999 Feb 26;274(9):5429-35. Cotten JF, Welsh MJ
Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge.
J Biol Chem. 1999 Feb 26;274(9):5429-35., 1999-02-26 [PMID:10026154]

Abstract [show]
Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wild-type CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction.

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No. Sentence Comment
154 We studied the conductance properties of the following double mutants: R347D/D924R, R347D/D993R, and R347E/E1104R. Login to comment
155 The R347D/D993R and R347E/E1104R mutants each had two conductance states with pHc-dependent behavior (Fig. 5). Login to comment
156 For R347D/D993R the increased entry into the OB state was apparent as a shoulder on the amplitude histogram at pHc 5.5. Login to comment
157 Accordingly, for R347D/D993R and R347E/E1104R the current variance in the open state increased with decreasing pHc (Fig. 5B). Login to comment
158 Qualitatively, the lifetimes of the OL and OB conductance states in the R347D/D993R and R347E/E1104R were similar to that of the R347D and R347E mutants, respectively. The amplitude of the OL state was larger for both of these double mutants as compared with the single mutants (Figs. Login to comment
179 A, single-channel current tracings from excised, inside-out membrane patches containing R347E/E1104R, R347D/D924R, and R347D/D993R. Login to comment
181 B, current variance of R347E/E1104R, R347D/D924R, and R347D/D993R at the indicated pHc was collected as in Fig. 3. Login to comment
209 Consistent with this, mutation of D993R and E1104R in MSD2 increased the relative amplitude of the OL conductance state in the context of Arg-347 mutations. Login to comment

[hide] Mutations at arginine 352 alter the pore architect... J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18. Cui G, Zhang ZR, O'Brien AR, Song B, McCarty NA
Mutations at arginine 352 alter the pore architecture of CFTR.
J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18., [PMID:18421494]

Abstract [show]
Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

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8 Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Login to comment
9 Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+ , while wild-type and R352E/D993R-CFTR were not. Login to comment
162 Slope conductances are summarized in Table 1 Table 1 Slope conductancea (in pS) of the f state of WT-CFTR and multiple single and double mutants CFTR n Negative VM Positive VM WT 7 6.82 ± 0.03 6.97 ± 0.06 R352A 6 6.80 ± 0.06 7.85 ± 0.07*, ** R352Q 6 5.29 ± 0.02* 6.28 ± 0.05*, ** R352K 5 6.87 ± 0.03 6.86 ± 0.01 R352E 5 3.78 ± 0.01* 6.03 ± 0.01*, ** R352E/E873R 6 3.84 ± 0.01* 5.64 ± 0.01*, ** R352E/ E1104R 6 4.36 ± 0.01* 5.86 ± 0.02*, ** R352E/D993R 5 5.90 ± 0.02* 6.44 ± 0.01*, ** D993R 7 8.27 ± 0.05* 7.13 ± 0.07** a Slope conductance indicates single-channel conductance calculated from 0 to +100 mV (positive VM) or to -100 mV (negative VM) by linear regression * P B 0.001 compared to the equivalent slope conductance in WT-CFTR, ** P B 0.001 compared to the slope conductance in the same mutant at negative VM reflects the loss of anion binding properties within the core of the permeation pathway, which contributes to the tight binding of SCN (Smith et al. 1999). Login to comment
166 Our present results suggest -300 -50 300 50 Br -100 100 NO3 Cl SCN pA mVBr NO3 SCN Cl -300 -50 300 50 Br -100 100 NO3 Cl SCNC pA mVBr NO3 SCN Cl R352E -4000 -50 4000 50 -100 100 -800 -50 800 50 -100 100 -6000 -50 6000 50 -100 100 A pA -50 800 50 -100 100 A SCN Br Cl NO3 NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl -800 mV pA mV pA mV pA mV NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl Br NO3 SCN Cl -4000 -50 4000 50 -100 100 D -800 -50 800 50 -100 100 E D993R -6000 -50 6000 50 -100 100 WT pA -50 800 50 -100 100 B SCN Br Cl NO3 NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl -800 mV pA mV pA mV pA mV NO3 Br Cl SCN Br NO3 SCN Cl Br NO3 SCN Cl Br NO3 SCN Cl R352A R352K R352E/ Fig. 5 Mutations at R352 alter anion selectivity. Login to comment
167 Representative inside-out macropatches, recorded in the presence of cytoplasmic Cl- or Cl- plus substitute anions, with voltage ramps between -100 and +100 mV, are shown for (A) WT-CFTR, (B) R352A-CFTR, (C) R352E-CFTR, (D) R352K-CFTR and (E) the double mutant R352E/D993R-CFTR. Login to comment
171 Solutions were at pH 7.45 and are labeled as follows: 150 mM Cl- (black), 130 mM Cl- plus 20 mM NO3 - (purple), 130 mM Cl- plus 20 mM Br- (green) and 130 mM Cl- plus 20 mM SCN- (red) Table 2 Relative permeabilities of some anions in WT-CFTR and R352-CFTR mutants * Significant difference compared with WT-CFTR, P \ 0.05; ** Significant difference compared with R352A, P \ 0.05 CFTR n SCN Br NO3 WT 6 4.11 ± 0.17 1.45 ± 0.04 1.51 ± 0.02 R352A 10 4.18 ± 0.65 1.35 ± 0.21 1.70 ± 0.29 R352E 6 5.18 ± 0.32* 1.47 ± 0.08 1.64 ± 0.43 R352K 7 4.05 ± 0.12 1.52 ± 0.01 1.59 ± 0.03** R352E/D993R 6 3.62 ± 0.06* 1.48 ± 0.04 1.59 ± 0.02** Table 3 Relative conductances of some anions in WT-CFTR and R352-CFTR mutants CFTR n SCN Br NO3 WT 6 0.16 ± 0.02 0.67 ± 0.04 0.84 ± 0.04 R352A 10 1.59 ± 0.12* 1.31 ± 0.08* 1.59 ± 0.14* R352E 6 2.73 ± 0.31*, ** 1.49 ± 0.22* 1.54 ± 0.12* R352K 7 1.12 ± 0.08*, ** 0.99 ± 0.02*, ** 1.73 ± 0.26* R352E/ D993R 7 0.61 ± 0.05*, ** 0.98 ± 0.03*, ** 1.26 ± 0.13* Relative conductance was measured at VM = Vrev -25 mV * Significant difference compared with WT-CFTR, P\0.05; ** Significant difference compared with R352A, P\0.05 that loss of positive charge at position 352 destroyed the overall pore architecture, which subsequently changed the anion selectivity characteristics as seen in R352A- and R352E-CFTR. Login to comment
199 We studied the conductance properties of CFTR channels bearing the following mutations: R352E, R352E/E873R, R352E/ D993R and R352E/E1104R. Login to comment
202 R352E/D993R-CFTR, in contrast, exhibited stability of the full conductance state similar to that seen in WT-CFTR and R352K-CFTR (Fig. 1); transitions to the s1 and s2 states were rare events in this double mutant. Login to comment
205 The slope conductance of R352E/D993R-CFTR was slightly lower than that of WT-CFTR, although linearity of the i-V relation was mostly retained. Login to comment
206 These data suggested that the D993R mutation at least partly compensated for the R352E mutation, although the double mutant R352E/ D993R-CFTR did not fully recapitulate the behavior of WT-CFTR. Login to comment
208 If D993 served as the interaction partner of R352, we would expect that block of R352E/D993R-CFTR would be similar to that 0.4 pA 2 s 0.4 pA 2 s 0.2 pA 2 s 0.2 pA 2 s c s1 s2 f c s1 s2 f c s1 s2 f c f R352E R352E/E873R R352E/E1104R R352E/D993R 0 4000 #ofevents 0.0 -0.4 -0.8 0.0 -0.4 -0.8 3000 #ofevents 0 #ofevents 0.0 -0.4 -0.8 3000 0 Currents (pA) 0.0 -0.4 0 2500#ofevents -0.8 fc s1 s2 s1 s2 s1 s2 0.4 pA 2 s 0.4 pA 2 s 0.2 pA 2 s 0.2 pA 2 s c s1 s2 f c s1 s2 f c s1 s2 f c f R352E R352E/E873R R352E/E1104R R352E/D993R 0.4 pA 2 s 0.4 pA 2 s 0.4 pA 2 s 0.4 pA 2 s 0.2 pA 2 s 0.2 pA 2 s 0.2 pA 2 s 0.2 pA 2 s c s1 s2 f c s1 s2 f c s1 s2 f c f R352E R352E/E873R R352E/E1104R R352E/D993R B C D A 0 4000 #ofevents 0.0 -0.4 -0.8 0 4000 #ofevents 0.0 -0.4 -0.8 0.0 -0.4 -0.8 3000 #ofevents 0 0.0 -0.4 -0.8 3000 #ofevents 0 #ofevents 0.0 -0.4 -0.8 3000 0 Currents (pA) #ofevents 0.0 -0.4 -0.8 3000 0 #ofevents 0.0 -0.4 -0.8 3000 0 Currents (pA) 0.0 -0.4 0 2500#ofevents -0.8 fc s1 s2 s1 s2 s1 s2 Fig. 7 Single-channel current tracings of R352E-CFTR and double mutants from excised inside-out patches (left) and resulting all-points amplitude histograms (right) under the same experimental conditions as in Fig. 1. Login to comment
210 There are four current levels indicating the c, s1, s2 and f states in all but the revertant mutant R352E/ D993R-CFTR, which only exhibited the c and f states. Login to comment
213 Figure 8B shows macropatch currents from R352E/D993R-CFTR in the presence and absence of 200 lM glipizide; time-dependent block was rescued in this double mutant. Login to comment
215 This result suggested that the revertant double mutation, R352E/ D993R, recovered the time-dependent block by glipizide but did not completely recover the sensitivity to glipizide characteristic of WT-CFTR. This may reflect the difference in side chain volumes between aspartic and glutamic acids; the volume of a glutamic acid side chain is 20% larger than that of an aspartic acid side chain (Creighton 1993), which may result in a different pore structure. Login to comment
216 To further explore the characteristics of R352E/D993R-CFTR, we studied anion selectivity between Cl- and four substitute monovalent anions. Login to comment
218 Overall, both relative permeability and relative conductance values for WTand R352E/ D993R-CFTR were similar (Tables 2, 3). Login to comment
219 R352E/D993R- CFTRcurrentsexhibitedthesameanionpermeabilitysequence as WT-CFTR: SCN- [NO3 - C Br- [Cl- (although PSCN/ PCl was clearly reduced in the double mutant). Login to comment
220 R352E/D993R-CFTR exhibited relative conductances to SCN- and Br- intermediate between that of WT-CFTR and R352A-CFTR (Table 3). Login to comment
221 These results also suggested that R352A-CFTR and R352E/D993R-CFTR have different pore architecture and that the selectivity properties of the pore of the double mutant might be slightly different from that of WT-CFTR. Login to comment
222 The D993R Mutation Alone also Altered the Pore Architecture of CFTR D993 is localized to TM9 of CFTR, which has not been suggested to be a pore lining domain (McCarty 2000). Login to comment
223 Because the data presented thus far suggested that D993 serves as the interacting partner of R352, we predicted that the D993R mutation alone would change channel activity in a manner similar to that of the R352E, -A or -Q mutation. Login to comment
224 We studied D993R-CFTR with single-channel recording techniques using the same conditions as in Fig. 1. Login to comment
225 D993R-CFTR exhibited instability of the open state, with frequent transitions between all three open conductance levels (Fig. 9A, B); these three open states were even less stable than those of R352A-CFTR. Login to comment
226 The slope conductance of the f state in D993R-CFTR was larger than that of WT-CFTR and indicated slight inward rectification (Fig. 9C, Table 1). Login to comment
227 These results suggest that the D993R mutation alone also destroyed pore architecture in a manner similar to that of the charge-destroying mutations at R352. Login to comment
232 Hence, it is likely that MTSEA+ modified one (or more) of the endogenous cysteines, which B WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA 100 ms 0.2 nA A WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R WT-CFTR R352E/D993R R352E R352E/E873R R352E/E1104R mV -100 -50 50 100 -0.8 -0.4 0.4 0.8 pA 100 ms 0.2 nA Fig. 8 The double mutant R352E/D993R-CFTR recovers WT-like channel behavior. Login to comment
233 (A) Single channel i-V relationships are shown for full conductance states of WT-, R352E-, R352E/E873R-, R352E/ E1104R- and R352E/D993R-CFTR. Login to comment
235 (B) Block of R352E/ D993R-CFTR macropatch currents by glipizide (200 lM) is time-dependent. Login to comment
239 If this were true, we would expect that the revertant double mutant, R352E/D993R-CFTR, would not respond to MTSEA+ . Login to comment
244 In contrast, R352E/ D993R-CFTR was insensitive to exposure to MTSEA+ , which resulted in only a 1.08 ± 0.02-fold increase in current (n = 6), thus indicating that the double mutant exhibited WT-like behavior. Login to comment
250 These results strongly suggested that the loss of the positively charged side chain at position 352 shifted the pore architecture in such a way as to destabilize the full open state, 0.2 pA 2 s c s1 s2 f 0.2 pA 2 s c s1 s2 f Currents (pA) 0.0 -0.4 -0.8 2000 4000 6000 #ofevents mV -100 -50 50 100 -1.0 -0.5 0.5 1.0 WT-CFTR D993R pA 0.2 pA 2 s c s1 s2 f 0.2 pA 2 s c s1 s2 f A CB Currents (pA) 0.0 -0.4 -0.8 2000 4000 6000 #ofevents Currents (pA) 0.0 -0.4 -0.8 2000 4000 6000 #ofevents mV -100 -50 50 100 -1.0 -0.5 0.5 1.0 WT-CFTR D993R pA mV -100 -50 50 100 -1.0 -0.5 0.5 1.0 WT-CFTR D993R WT-CFTR D993R pA Fig. 9 Mutation D993R alone had effects similar to those of the charge-reversing mutations at R352. Login to comment
251 Representative single-channel current tracing (A), all-points amplitude histogram (B) and i-V relationship for the f conductance state (C) are shown for the D993R mutant. Login to comment
253 In c, points show mean ± SEM for n = 7 observations, and error bars are smaller than the symbols; lines are from linear regression WT 1 A 200 s Isoproterenol 0.4 A 100 s 0.4 A 100 s R352A R352E/D993R MTSEA MTSEA MTSEA WT 1 A 200 s Isoproterenol 0.4 A 100 s 0.4 A 100 s R352A R352E/D993R MTSEA MTSEA MTSEA Fig. 10 Mutation R352A results in appearance of sensitivity to a cysteine-modifying reagent. Login to comment
254 Oocytes expressing WT-CFTR (top trace), R352A-CFTR (middle trace) or R352E/D993R-CFTR (bottom trace), along with the b2-adrenergic receptor, were studied by two-electrode voltage clamp. Login to comment
258 Second, we identified the interaction partner as D993 by use of double mutants; R352E/E873R-CFTR and R352E/E1104R-CFTR exhibited permeation properties similar to those of R352E-CFTR, while R352E/D993R-CFTR behaved more like WT-CFTR. Login to comment
261 As predicted, D993R-CFTR exhibited instability of the open state similar to that seen in R352E-CFTR. Login to comment
295 Stability of the open state was retained in the case of a charge-conserving mutation, R352K, and in the double mutant R352E/D993R-CFTR. Login to comment
297 Compared to WT-CFTR, R352E/D993R-CFTR channels exhibited lower slope conductance, weakened block by glipizide, and altered selectivity between Cl- and SCN- . Login to comment
301 We conclude that the double mutant R352E/D993R-CFTR retains the interaction between these residues but does not fully mimic the behavior of WT-CFTR, suggesting that permeation properties in the CFTR chloride channel are very sensitive to small changes in pore structure. Login to comment

[hide] Evolutionary and functional divergence between the... Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18865-70. Epub 2008 Nov 19. Jordan IK, Kota KC, Cui G, Thompson CH, McCarty NA
Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters.
Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18865-70. Epub 2008 Nov 19., 2008-12-02 [PMID:19020075]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.

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No. Sentence Comment
95 Isolated bursts of channel activity from oocytes expressing WT-CFTR, R352E-CFTR, R352E/E1104R-CFTR, and R352E/D993R-CFTR. Login to comment
100 In contrast, WT-CFTR channels, and channels bearing the revertant mutation R352E/D993R, primarily exhibit transitions to the full conductance level. Login to comment
131 In contrast, approximately wild-type channel behavior is retained in R352K-CFTR and the charge-swapping double mutant, R352E/D993R-CFTR (Fig. 3). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2009 Oct 27;48(42):10078-88. Alexander C, Ivetac A, Liu X, Norimatsu Y, Serrano JR, Landstrom A, Sansom M, Dawson DC
Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore.
Biochemistry. 2009 Oct 27;48(42):10078-88., 2009-10-27 [PMID:19754156]

Abstract [show]
The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

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No. Sentence Comment
306 Conversely, Cui et al. (41) reported that substitution of a positive charge at position 993 (D993R) increased single-channel conductance for inward current above that of wt CFTR. Login to comment

[hide] Differential contribution of TM6 and TM12 to the p... Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13. Cui G, Song B, Turki HW, McCarty NA
Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers.
Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13., [PMID:22160394]

Abstract [show]
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 muM Glyb and 200 muM Glip was 0.64 +/- 0.03 (n = 7) and 0.48 +/- 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 muM Tolb was 0.40 +/- 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.

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No. Sentence Comment
144 These functional characteristics were returned to approximately their wildtype behavior in R352E/D993R-CFTR, perhaps because the salt bridge was retained in this second-site reversion [12]. Login to comment

[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]

Abstract [show]
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.

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No. Sentence Comment
21 However, subconductance states are dominant events with short burst durations in CFTR channels bearing known salt bridge mutations, such as R352A, R347H, D993R, and D924R (13, 14). Login to comment
105 Similar results were found for D993R-CFTR, but nearly wild type-like behavior, including stable openings to the f state, was recovered in the R352E/D993R double mutant (see Fig. 3A). Login to comment
109 We therefore hypothesized that Arg347 might also interact with Asp993 to rescue the CFTR channel pore to a stable f state and tested this hypothesis in three double mutants; TABLE 1 Summary of the effects of mutations studied Mutant Main features of open bursts Impact on f state R347A Emphasizes s1 state, brief transitions to s2 and f Can reach f but not stable R347D Emphasizes s1 state, no transitions to s2 and f Cannot reach f D924R Brief transitions to all conductance levels Can reach f but not stable R347K Wild type-like Wild type-like R347D/D924R Emphasizes s2 state, rare and brief transitions to f Can reach f but not stable R352E Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable D993R Opens to all 3 levels, but none are stable Can reach f but not stable R352E/D993R Wild type-like, with increased transitions to s1 and s2; slightly reduced single-channel conductance Wild type-like R352E/D924R Opens to all 3 levels, but none are stable Can reach f but not stable R347D/D993R Very stable s2; rare and brief transitions to both s1 and f Can reach f but not stable R347A/R352A Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable R347D/D924R/D993R Opens to all 3 levels; s1 much more stable than in WT, s2 relatively stabilized, f unstable Can reach f but not stable R347D/D924R/R352E/D993R Primarily flickers between s2 and f; s1 much more stable than in WT, slightly reduced single channel conductance Can reach f but not stable FIGURE 3. Login to comment
111 A, representative current samples of R352E/D993R-, R352E/D924R-, and R347D/D993R-CFTR recorded from excised inside-out patches with the same conditions as Fig. 2 (n afd; 3-6 for each mutant). Login to comment
114 As noted above, R352E/D993R exhibited a prominent full open state similar to WT-CFTR (13), suggesting that the R352E/D993R salt bridge can fully rescue the CFTR channel pore to normal behavior (aside from a slight decrease in single channel conductance). Login to comment
115 Whereas the single channel behavior of R352E/D924R was similar to that of R352E alone, with multiple unstable open states, suggesting that Arg352 and Asp924 do not interact, R347D/D993R was much more like R347D/D924R, with the s2 state dominant (compare Figs. 3 and 2). Login to comment
116 R347D/ D993R-CFTR is able to transition to the f state but sojourns there are even more brief than those seen for the R347D/ D924R. Login to comment
122 Similarly, in the R347D/D993R mutant, the positive charge at Arg347 is no longer available to interact with Asp924 . Login to comment
124 This was tested in the triple mutant R347D/D924R/D993R (Fig. 4, A and B). Login to comment
125 Unlike the two double mutants described above (R347D/D924R and R347D/ D993R), the triple mutant exhibited roughly equal occupancy of s1,s2,andfstates;theoccupancyofthes2statewasnotasstableas in either double mutant. Login to comment
141 However, the quadruple mutant R347D/D924R/D993R/R352E did not completely rescue WT behavior (Fig. 4, A and B). Login to comment
146 Representative current samples of R347A/R352A-, R347D/D924R/D993R-, and R347D/D924R/D993R/R352E-CFTR were recorded under the same conditions as in Fig. 3 (n afd; 5-6 for each mutant) (A). Login to comment