ABCMdb

ABCC7 p.Arg352Glu

ClinVar:	c.1054C>T , p.Arg352Trp D , Likely pathogenic c.1055G>A , p.Arg352Gln D , Pathogenic
CF databases:	c.1055G>A , p.Arg352Gln D , CF-causing ; CFTR1: This missense mutation, at nucleotide position 1187 (G to A) in exon 7, has been detected in an Italian CF patient through DGGE and direct sequencing. The mutation generates an Arg to Gln substitution (R352Q) and creates a novel DdeI restriction site in the mutated allele. This mutation has been detected in a PS patient (paternal chromosome), associated with the haplotype A; the maternal chromosome carries a still uncharacterized mutation. It was found in one of 60 non-[delta] Italian CF chromosomes. c.1054C>G , p.Arg352Gly (CFTR1) ? , c.1054C>T , p.Arg352Trp (CFTR1) ? , The mutation was detected by SSCP/heteroduplex analysis and identified by direct DNA sequencing. The mutation was seen in a boy referred by West Midlands Regional Genetics Service, and whose other CF mutation was [delta]F508. We have seen it only once in over 150 samples tested.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (91%), K: D (85%), L: D (91%), M: D (95%), N: D (95%), P: D (95%), Q: D (59%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Arg352 is a major determinant of charge selectivit... Biochemistry. 1999 Apr 27;38(17):5528-37. Guinamard R, Akabas MH
Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel.
Biochemistry. 1999 Apr 27;38(17):5528-37., 1999-04-27 [PMID:10220340]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We investigated the role of Arg352, a residue flanking the predicted cytoplasmic end of the M6 segment, in the mechanism of charge selectivity. We determined the Cl- to Na+ permeability ratio (PCl/PNa) from the reversal potential measured in a 10-fold NaCl gradient. For the wild type, PCl/PNa was 36 (range of 28-51). For the R352H mutant, PCl/PNa was dependent on cytoplasmic pH. At pH 5.4, the PCl/PNa was 33 (range of 27-41), similar to that of the wild type, but at pH 7.2, where the histidine should be largely uncharged, PCl/PNa was 3 (range of 2.9-3.1). For the R352C and R352Q mutants, PCl/PNa was 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Furthermore, Na+ which does not carry a significant fraction of the current through the wild type is measurably conducted through R352Q. Thus, the charge of the side chain at position 352 is a strong determinant of charge selectivity. In the wild type, the positive charge on Arg352 contributes to an electrostatic potential in the channel that forms a barrier to cation permeation. Mutation of Arg352 did not alter the halide selectivity sequence. Selectivity among halides must involve other residues.

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166 Unfortunately, the single-channel conductance and kinetics of the R352E mutant appeared to be too small to accurately determine the reversal potential. Login to comment

[hide] Positive charges at the intracellular mouth of the... J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16. Aubin CN, Linsdell P
Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.
J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16., [PMID:17043152]

Abstract [show]
Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl(-) ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl(-) ion permeation through the pore.

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7 However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl-concentration. Login to comment
43 In contrast to wild-type CFTR, mutants R303E and, to a lesser extent, R352E showed outward rectification of the I-V relationship with symmetrical 154 mM Cl- solutions. Login to comment
63 In contrast, both R303E and R352E were associated with strong outward rectification (Fig. 2 A), which is quantified in Fig. 2 B. Login to comment
78 Unfortunately, because of low current density in this mutant, R352E could not be studied at low Cl-concentration. Login to comment
95 (B) Mean single channel current-voltage relationships constructed from such recordings for wild type (■), R303E (●) and R352E (○). Login to comment
100 Single channel currents carried by R303E and R352E under the same symmetrical Cl- solutions used in Fig. 2 are shown in Fig. 5 A. Login to comment
103 For example, at -80 mV, single channel current amplitude was reduced by 89 ± 0% (n = 4) in R303E and by 75 ± 2% (n = 3) in R352E, while at +80 mV, unitary currents were reduced by 30 ± 1% (n = 4) in R303E and by 25 ± 1% (n = 4) in R352E (Fig. 5 B). Login to comment
104 The Cl-dependence of I-V shape in R303E and R352E is consistent with removal of positive surface charges that normally act to increase the Cl-concentration at the inner mouth of the pore. Login to comment
114 (A) Example single channel currents recorded from wild type, R303E, and R352E at different intracellular Cl- concentrations (154 or 304 mM, as indicated), at a membrane potential of -20 mV. For each trace, the line to the left represents the current level when the channel is closed. Login to comment
149 Under these conditions, current carried not only by wild-type CFTR, but also by each of the channel mutants R303E, R352E (Fig. 10), R80E, R242E, R933E, R1102E, and R352Q (not depicted) showed reversal potentials that were not significantly different from the calculated Cl- equilibrium potential (+33.4 mV). Login to comment
184 Furthermore, rectification in R303Q and R303E appeared more sensitive to the intracellular Cl-concentration than in R352Q and R352E (Fig. 3 B). Login to comment
196 Unfortunately we were unable to obtain single channel recordings at Cl- concentrations higher than 304 mM; however, the dramatic increase in conductance seen in both R303E and R352E when intracellular [Cl-] was increased from 154 to 304 mM (Fig. 6) suggests that the conductance of these mutant channels is far from saturated at this elevated Cl-concentration, consistent with a large increase in Km. Login to comment
199 Nevertheless, unitary currents carried by Cl- influx were also reduced in both R303E and R352E (Fig. 5). 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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No. Sentence Comment
6 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. Login to comment
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
137 Both f state slope conductances were significantly reduced in R352E-CFTR (Table 1). Login to comment
139 The single-channel conductance of the f state exhibited significant outward rectification in R352A-, R352Q- and R352E-CFTR (Table 1). Login to comment
146 Anion Selectivity of R352A-, R352E- and R352K-CFTR To determine whether mutations at R352 affected the ability of CFTR channels to select between ions of similar charge, we studied the anion selectivity patterns of R352A-, R352E- and R352K-CFTR using inside-out macropatches and compared them to WT-CFTR. Login to comment
152 For calculation of Gx/GCl, we compared R352A-, R352E- and R352K-CFTR with WT-CFTR as well as R352E- and R352K-CFTR with R352A-CFTR. 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
173 Furthermore, the finding that relative permeability values are nearly identical in R352A-, R352E- and R352K-CFTR suggests that the role of this site in determining anion selectivity is only indirect. Login to comment
186 In summary, mutations at R352 that destroyed the positive charge (R352A, R352E and R352Q) altered the pore architecture of CFTR and caused instability of the open state, changing anion selectivity and pore block by glipizide. Login to comment
198 To identify the interaction partner for R352, we replaced R352 with an acidic residue (R352E) and introduced an arginine residue in the place of candidate interaction partners. 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
201 Three of these mutants, R352E-, R352E/E873R- and R352E/E1104R-CFTR, exhibited instability of the open state, in which the amplitudes of the s1, s2 and f conductance states were very similar between the three mutants. 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
204 R352E-, R352E/ E873R- and R352E/E1104R-CFTR exhibited significant outward rectification, while WT-CFTR did not (Table 1). 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
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
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
246 First, channels bearing charge-destroying mutations at this site, including R352Q, R352E and R352A, exhibited instability of the open state compared to WT-CFTR, as indicated by frequent transitions between all three open conductance states (s1, s2, f). 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
300 We also note that while the relative conductance values for SCN- , Brand NO3 - are shifted in the same direction in R352K-CFTR as they are in R352A- or R352E-CFTR, the shifts for SCN- and Br- are smaller in R352K-CFTR than in the charge-destroying mutants. 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
99 Channels bearing the R352E mutation, or the double mutant R352E/E1104R, exhibited frequent transitions to subconductance levels. 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] 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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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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104 In our prior studies, we found that R352E-CFTR can open to all three conductance levels, with all open states being unstable. 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
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
162 Recovery of Charge at R352C and D993C Rescued Channel Stability in the Full Open State-R352C-CFTR exhibited single channel behavior similar to that previously reported for R352A-, R352Q-, and R352E-CFTR (13). Login to comment

[hide] Acute inhibition of the cystic fibrosis transmembr... Am J Physiol Cell Physiol. 2013 Oct 15;305(8):C817-28. doi: 10.1152/ajpcell.00052.2013. Epub 2013 Jun 19. Cai Z, Li H, Chen JH, Sheppard DN
Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms.
Am J Physiol Cell Physiol. 2013 Oct 15;305(8):C817-28. doi: 10.1152/ajpcell.00052.2013. Epub 2013 Jun 19., [PMID:23784545]

Abstract [show]
The chemical structures of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We therefore tested the acute effects of T3, T4 and reverse T3 (rT3) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T3, T4, and rT3 (all tested at 50 muM) inhibited CFTR in several ways: they strongly reduced CFTR open probability by impeding channel opening; they moderately decreased single-channel current amplitude, and they promoted transitions to subconductance states. To investigate the mechanism of CFTR inhibition, we studied T3. T3 (50 muM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T3 was weakly voltage dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T3 inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to subconductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to subconductance states. We conclude that T3 has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.

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251 The occurrence of subconductance states is more frequent in some site-directed mutations in the MSDs [e.g., R334C-CFTR (70), R347E-CFTR (11), and R352E-CFTR (13)], while wild-type murine CFTR resides for prolonged periods in a minuscule subconductance state and only transitions infrequently to the full open-state (37). Login to comment