ABCMdb

PMID: 18366345

Caci E, Caputo A, Hinzpeter A, Arous N, Fanen P, Sonawane N, Verkman AS, Ravazzolo R, Zegarra-Moran O, Galietta LJ
Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis.
Biochem J. 2008 Jul 1;413(1):135-42., 2008-07-01 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg347Ala We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTRinh-172 by 20-30-fold. Login to comment 24 ABCC7 p.Gly551Asp ABCC7 p.Pro574His ABCC7 p.Gly1349Asp However, NBD mutants such as G551D, G1349D or P574H, do not show an altered sensitivity to CFTRinh-172 [9,12]. Login to comment 25 ABCC7 p.Gly551Asp In contrast, the potency of CFTR activators, whose effect involves direct interaction with the NBDs, is greatly altered by mutations such as G551D which resides in NBD1 [13-15]. Login to comment 84 ABCC7 p.Arg347Ala Experiments were carried out on FRT cells with stable expression of wild-type or R347A CFTR. Login to comment 97 ABCC7 p.Arg347Ala Figure 1 Mutants of the sixth TMD (A) Representative traces showing normalized cell fluorescence recordings and quenching upon I- addition in COS-7 cells transfected with wild-type (top panel) or R347A (bottom panel) CFTR. Login to comment 101 ABCC7 p.Arg352Ala Wild-type CFTR and all mutants except R352A showed an I- influx significantly higher (P < 0.01) than mock-transfected cells. Login to comment 110 ABCC7 p.Thr338Ala ABCC7 p.Arg347Ala T338A and R347A were similar to wild-type CFTR. Login to comment 111 ABCC7 p.Ser341Ala ABCC7 p.Arg334Ala R334A and S341A showed reduced anion transport, although this was significantly greater than cells transfected with the fluorescent protein alone (Figure 1B). Login to comment 112 ABCC7 p.Arg352Ala More marked was the effect of R352A, which showed undetectable levels of CFTR activity. Login to comment 114 ABCC7 p.Arg352Ala The absence of function did not allow us to measure the potency of CFTRinh-172 for the R352A mutant. Login to comment 118 ABCC7 p.Arg347Ala The exception was R347A. Login to comment 124 ABCC7 p.Arg347Ala **Indicate that the R347A Ki was significantly higher (P < 0.01) compared with wild-type CFTR. Login to comment 125 ABCC7 p.Arg347Ala For Arg347 mutants other than R347A, sensitivity to CFTRinh-172 was so low that the Ki could not be determined precisely (see Experimental section). Login to comment 127 ABCC7 p.Arg347Cys ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Arg334Ala ABCC7 p.Arg347Ala ABCC7 p.Ala349Ser CFTR form CFTRinh-172 Ki (μM) Hill coefficient I- influx (mM/s) n Wild-type 1.32 + - 0.25 1.03 + - 0.07 0.1336 + - 0.0107 10 S341A 0.57 + - 0.17 1.21 + - 0.37 0.0297 + - 0.0064 4 T338A 3.20 + - 0.86 1.13 + - 0.20 0.1260 + - 0.0225 4 R347A 44.98 + - 4.71** 0.91 + - 0.04 0.1288 + - 0.0154 7 R334A 2.39 + - 0.74 0.93 + - 017 0.0313 + - 0.062 4 A349S 1.23 + - 0.41 1.11 + - 0.25 0.1500 + - 0.011 4 R347D >50 Not determined 0.1160 + - 0.0136 7 R347D/D924R >50 Not determined 0.1008 + - 0.0504 4 R347C >50 Not determined 0.1437 + - 0.0123 4 Mock 0.003 + - 0.001 10 introduced a mutation at position 349 (an alanine residue replaced by a serine residue). Login to comment 129 ABCC7 p.Ala349Ser The A349S mutant had anion transport ability (Figure 1B) and sensitivity to CFTRinh-172 (Ki = 1.23 +- 0.41 μM, Table 1) similar to those of the wild-type channel. Login to comment 132 ABCC7 p.Arg347Cys ABCC7 p.Arg347Asp ABCC7 p.Arg347Ala As found for R347A, the mutants R347C and R347D also showed a normal rate of anion transport but altered sensitivity to CFTRinh-172 (Figures 2A and 2B). Login to comment 134 ABCC7 p.Asp924Arg ABCC7 p.Asp924Ala Because Arg347 is believed to be involved in the formation of a salt bridge with Asp924 [25], we also tested the CFTR mutants D924A and D924R. Login to comment 136 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg To further investigate the importance of the salt bridge, we generated a double mutant, R347D/D924R, in which the positions of the charged amino acids are inverted. Login to comment 137 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg Interestingly, in contrast with D924R, the double mutant was able to transport anions but showed a low sensitivity to CFTRinh-172, with an estimated Ki greater than 50 μM (Figures 2A and 2B), similar to that of the single R347D mutant. Login to comment 138 ABCC7 p.Asp924Arg We evaluated the maturation of the D924R mutant by immunoprecipitation followed by Western blot analysis or in vitro phosphorylation (Figure 2C). Login to comment 139 ABCC7 p.Arg347Asp Wild-type protein and the R347D mutant showed a normal pattern of electrophoretic mobility with a prevalent abundance of the band C which represents the fully glycosylated mature form of CFTR. Login to comment 141 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg In contrast with R347D, the D924R mutation produced a partial defect in maturation, with increased band B intensity compared with band C. Interestingly, this defect seemed to be corrected in the double mutant R347D/D924R (Figure 2C). Login to comment 143 ABCC7 p.Arg347Asp ABCC7 p.Arg334Ala ABCC7 p.Arg347Ala FRT cells were stably transfected with wild-type, R334A, R347A and R347D CFTR, and transepithelial Cl- currents were measured. Login to comment 144 ABCC7 p.Arg347Asp ABCC7 p.Arg347Ala The R347A and R347D mutants showed Figure 2 Mutagenesis of Arg347 and Asp924 residues (A) Rate of I- transport measured in COS-7 cells transfected with the indicated constructs. Login to comment 145 ABCC7 p.Asp924Arg ABCC7 p.Asp924Ala In contrast with all other constructs, D924A and D924R did not show a significant I- transport compared with mock-transfected cells. Login to comment 146 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg (B) CFTRinh-172 dose-response relationships for wild-type, R347D and R347D/D924R CFTR. Login to comment 155 ABCC7 p.Arg347Asp ABCC7 p.Arg347Ala The calculated Ki for wild-type CFTR, R347A and R347D was 0.85 +- 0.13 μM (n = 8), 17.35 +- 3.90 μM (n = 13) and 53.10 +- 4.74 μM (n = 6) respectively (Figure 3E). Login to comment 157 ABCC7 p.Arg334Ala For comparison, the sensitivity of the R334A mutant was not significantly altered (Ki = 0.50 +- 0.18 μM, n = 6; Figures 3C and 3E), in agreement with the fluorescence assay results. Login to comment 158 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg334Ala ABCC7 p.Arg347Ala Interestingly, the R347D mutant, although insensitive to CFTRinh-172, was fully inhibited by the open-channel blocker GlyH-101 Figure 3 CFTR Cl- current inhibition by CFTRinh-172 (A-D) Representative traces showing recordings of transepithelial Cl- currents measured in FRT cells with stable expression of wild-type (WT), R347A, R334A and R347D-CFTR. Cells were first stimulated with 20 μM forskolin to activate CFTR and then tested with increasing concentrations of CFTRinh-172. Login to comment 163 ABCC7 p.Arg347Asp Dose-responses carried out on wild-type CFTR and R347D cells showed identical sensitivity to GlyH-101 with a Ki of 5.02 +- 0.90 μM (n = 6) and 4.99 +- 0.66 μM (n = 6) respectively (Figures 4A-4C). Login to comment 164 ABCC7 p.Arg347Asp We also tested the sensitivity of wild-type and R347D to a zwitterionic, net neutral analogue of CFTRinh-172, thiazo N-O, in which the carboxyphenyl group is replaced by an oxido-4-pyridinyl group (Figure 5A). Login to comment 165 ABCC7 p.Arg347Asp Although less potent than CFTRinh-172, the thiazo N-O compound caused a dose-dependent decrease of wild-type CFTR currents (Ki = 31.42 +- 7.30 μM, n = 4), but very weak inhibition of the R347D mutant (Figures 5B and 5C). Login to comment 169 ABCC7 p.Arg347Ala In cells expressing the R347A mutant (n = 6), cAMP stimulation elicited currents with a moderate outward rectification of the current-voltage relationship (Figures 6C and 6D). Login to comment 170 ABCC7 p.Arg347Asp Despite the use of a CFTRinh-172 concentration an order of magnitude higher than the half-effective concentration for wild-type CFTR, the Cl- currents Figure 4 CFTR Cl- current inhibition by GlyH-101 (A and B) Representative traces showing transepithelial Cl- currents measured in FRT cells with stable expression of wild-type (WT) and R347D-CFTR. Cells were first stimulated with 20 μM forskolin to activate CFTR and then were tested with increasing concentrations of GlyH-101. Login to comment 173 ABCC7 p.Arg347Ala of the R347A mutant were only partially inhibited (Figures 6C and 6D). Login to comment 175 ABCC7 p.Arg347Ala 50% reduction of R347A currents whereas the inhibition of wild-type currents at 5 μM was nearly total. Login to comment 190 ABCC7 p.Arg347Asp (B) Representative recordings showing effect of increasing concentrations of thiazo N-O on wild-type and R347D activity. Login to comment 200 ABCC7 p.Asp924Arg Indeed, D924R causes a reduced amount of fully glycosylated protein (band C) that may indicate that the mutant protein is less stable. Login to comment 202 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Arg347Ala Interestingly, when we generated the double mutant R347D/D924R, in which the positions of positive and negative charges are inverted but the salt bridge is maintained [25], we Figure 6 Patch-clamp analysis of CFTR inhibition by CFTRinh-172 (A and C) Superimposed membrane currents recorded from cells expressing wild-type and the R347A mutant at membrane potentials between -100 and +100 mV. Login to comment 203 ABCC7 p.Arg347Ala Currents were recorded in the absence and in the presence of CFTRinh-172 (5 μM for wild-type and 10 μM for R347A). Login to comment 205 ABCC7 p.Arg347Ala (E) Summary of block caused by CFTRinh-172 at all membrane potentials on wild-type (at 5 μM) and R347A (at 10 μM). Login to comment 206 ABCC7 p.Arg347Ala Values are means + - S.E.M. (n = 6 for both wild-type and R347A). Login to comment 208 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg On the other hand, we found that the double mutant R347D/D924R did not behave as the wild-type CFTR in terms of CFTRinh-172 sensitivity but was more similar to single Arg347 mutants. Login to comment 211 ABCC7 p.Arg347Asp ABCC7 p.Arg347Ala We hypothesized that the negatively charged carboxyl group in CFTRinh-172 interacts with the positive charge of Arg347 , such that the effects of Arg347 mutations could be explained by loss of electrostatic attraction (R347A) or generation of electrostatic repulsion (R347D). Login to comment 212 ABCC7 p.Arg347Asp Accordingly, the activity of the neutral thiazo N-O compound had to be less affected by the R347D mutation. Login to comment 213 ABCC7 p.Arg347Asp However, the potency of this compound on the R347D mutant was reduced, as found for CFTRinh-172, providing evidence against a direct electrostatic interaction between the CFTRinh-172 carboxyl group and Arg347 . Login to comment 219 ABCC7 p.Arg347Asp For example, the R347D mutation has been shown to alter ATPase activity in NBDs [35], a finding that points to strong conformational coupling between TMDs and NBDs. Login to comment 220 ABCC7 p.Arg347Asp However, it is important to point out that in the present study the R347D mutant, although being poorly inhibited by CFTRinh-172, showed an unaltered sensitivity to GlyH-101, an open-channel blocker acting from the extracellular side [8]. Login to comment