ABCMdb

ABCC7 p.Glu267Arg

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Publications

PMID: 23478129 [PubMed] Billet A et al: "CFTR: effect of ICL2 and ICL4 amino acids in close spatial proximity on the current properties of the channel."

No. Sentence Comment

72 In order to change the properties of the side chains, we replaced each amino acid by a small, uncharged residue (mutants E267A-CFTR and K1060A-CFTR) or by an oppositely charged residue (mutants E267R-CFTR, K1060E-CFTR). Login to comment
73 We also studied the double mutant E267R-K1060E-CFTR, where the negative and positive charges are inverted. Login to comment
86 100 200 -50 50 100 -100 100 100 100 200 C D B A I (pA/pF) I (pA/pF) I (pA/pF) V (mV) V (mV) V (mV) 5000pA 50ms E267R-K1060E E267A E267R K1060A K1060E E267A E267R wt E267R-K1060E K1060A K1060E ICL4 ICL2 K1060 E267 ICL1 ICL3 K1060 E267 ICL4 ICL2 ICL1 ICL3 % of maturation B C wt E267A E267R K1060A K1060E E267R-K1060E wt -50 50 100 -100 100 200 -50 50 100 -100 100 Fig. 2. Login to comment
93 Dotted lines represent a maturation similar to that of the wt protein (C, D) Whole cell chloride currents of HEK293 cells expressing wt-CFTR, E267A/R-, K1060A/Eor E267R-K1060E-CFTR mutants in presence of 10 bc;M Fsk. Login to comment
106 Next, replacement of a negatively charged side chain by a positive one (E267R mutant) induced a 90-95% reduction of the inward and outward Cl-currents calculated at negative or positive potentials (Fig. 2C-D). Login to comment
108 Finally, with the "inverted" double mutant (E267R-K1060E) the recorded Cl-current density was not significantly different from wt CFTR. Login to comment
140 Indeed, only substitution of the negatively charged residue E267 (E267R mutation) had a very drastic effect since it induced a complete loss of Cl-current. Login to comment
141 Finally, since the interchange of the two charged residues (E267R-K1060E) did not modify the chloride current density, one may hypothesize that the presence of an electrostatic interaction between the two amino acids is more critical than their respective positions. Login to comment

PMID: 25190805 [PubMed] Wang W et al: "An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening."

No. Sentence Comment

96 The charge-reversal mutations (E267R and K1060E) at these positions clearly had the most dramatic effects on CFTR activity (the E267K mutation also markedly inhibited CFTR activity; see Fig. 5). Login to comment
102 CFTR Gating Mechanism 30366 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, Fig. provides indirect but persuasive evidence that the E267R and K1060E charge-reversal mutations reduced channel activity primarily by reducing the rate of channel opening rather than by shortening the open channel bursts or decreasing the single channel conductance. Login to comment
103 Shown in Fig. 3, A and B are unitary current recordings of the E267R and K1060E mutants at FIGURE 1. Login to comment
124 These micropatch results indicate that the E267R and K1060E charge-reversal mutants have exceptionally low Pos under control and PKI-treated conditions (10-50-fold lower than WT-CFTR) primarily because they have very low channel opening rates, not abnormally brief open channel bursts. Login to comment
143 Here we combined one of these GOF mutations with the E267R and K1060E mutations to test: (i) if a GOF mutation could rescue the ATP-dependent channel activities of the E267 and K1060 charge-reversal mutants and (ii) if bundle formation and the predicted electrostatic interaction also facilitate channel opening in the absence of ATP binding or NBD dimerization. Login to comment
144 Regarding the first issue, Fig. 6A shows that introducing the K978C substitution into the E267R mutant substantially restored ATP-dependent channel activity (compare with Fig. 2B). Login to comment
145 The E267R/K978C double mutant exhibited normalized control currents and potentiator responses in macropatch experiments that were similar to wild type levels (see legend for mean data). Login to comment
154 B, macroscopic current record for patch containing many E267R-CFTR channels showing very small control current and very large activation by potentiators. Login to comment
156 C, macroscopic record showing that E267R-CFTR channels also are strongly stimulated by the FDA-approved potentiator, VX-770 (10 òe;M). Login to comment
171 Regarding the second issue, Fig. 6, B-E show that the E267R and K1060E mutations reduced the ATP-free activity of the K978C GOF mutant. Login to comment
174 The latter interpretation is further supported by the results in Fig. 7, which show that the E267R mutation also reduced the ATP-free channel activity of K978C/èc;1198-CFTR, a truncation mutant lacking NBD2 but possessing the indicated GOF mutation. Login to comment
176 Introducing the E267R mutation into this construct substantially reduced the basal ATP-independent currents and correspondingly increased the relative stimulation by potentiators (Fig. 7, B, D, and E). Login to comment
179 Combining a Catalytic Mutation with the E267R Mutation Rescues ATP-dependent Activity but Also Provides a Clue that R Domain Conformational Changes May Be Coupled to Bundle Formation-We next combined one of the charge-reversal mutations (E267R) with an NBD2 mutation (E1371S) that inhibits ATP hydrolysis at site 2 (Fig. 8). Login to comment
181 Our interest was to determine if the E267R mutation inhibited the activity of the E1371S catalytic mutant and/or destabilized the NBD dimer in the absence of ATP hydrolysis. Login to comment
182 Instead, however, the E267R/ E1371S-CFTR double mutant behaved similarly to the E1371S-CFTR single mutant with respect to: (i) high control currents in the presence of normally saturating ATP and PKA; (ii) negligible responses to potentiators presumably because the currents were already maximal under control conditions; and (iii) very slow deactivation upon ATP removal (঄deactivaton b0e; 5 min), indicative of a very tight NBD dimer (Fig. 8A; compare with FIGURE 3. Login to comment
183 Unitary current recordings indicate that the E267R and K1060E mutations do not obviously affect single channel conductance or open channel burst duration. Login to comment
184 A and B, micropatch recordings of E267R-CFTR and K1060E-CFTR channels obtained using smaller tip pipettes in which individual channel openings and closings and unitary currents were detectable before potentiator addition. Login to comment
195 We interpret these results to indicate that the E267R mutation has negligible effects on channel activity once a tight NBD dimer has been formed by ATP binding sans hydrolysis. Login to comment
200 Combining the E267R charge-reversal mutation with the E1371S mutation virtually abolished these PKA-independent currents (Fig. 8, B and D). Login to comment
202 E267R and K1060E Charge-reversal Mutations Strongly Reduce the PKA Sensitivity of Channel Gating-Fig. 9 shows that the E267R and K1060E mutations markedly impacted the PKA sensitivity of channel activation. Login to comment
247 Note that the E267K single mutation strongly inhibited channel activity as did the E267R mutation shown in Fig. 2. Login to comment
256 These authors observed greater inhibition by a charge-reversal mutation than by a neutral substitution at the E267 position (E267R versus E267A), as we showed here in our more detailed mechanistic study. Login to comment
260 Interestingly, Billet et al. (27) did report that a double mutant in which the charges were swapped between these sites (E267R/K1060E) behaved like wild type CFTR in their assay, which is consistent with an electrostatic interaction between these residues that impacts CFTR activity. Login to comment
262 Disrupting the E267-K1060 Interaction Primarily Impacts Channel Opening, not Burst Duration or NBD Dimer Stability-The charge-reversal mutations at these two positions dramatically decreased the macroscopic currents and apparent single FIGURE 6. Interplay between the charge-reversal mutations at the bundle interface and a previously reported GOF mutation (K978C); evidence that the E267RandK1060EmutationsalsoinhibitATP-freechannelactivity.A,E267R/K978CdoublemutantbehavesmorelikewildtypechannelsinthepresenceofATP inmacropatchexperiments.ConditionswereidenticaltoFig.2A.Themeanpercentcontrolcurrentsofthisdoublemutantwhennormalizedtothemaximalcurrents measuredafterpotentiatoradditionwere94afe;5.4%and82.8afe;3.9%beforeandafterPKIaddition,respectively(nafd;5).Thesevaluesarenotsmaller(infact,somewhat larger) than those for WT-CFTR (see Fig. Login to comment
264 B-D, macroscopic records for the indicated K978C single and double mutants showing that the E267R and K1060E substitutions decreased the fractional currents remaining after ATP removal. Login to comment
266 E267R/K978C-CFTR was activated strongly by the subsequent addition of 2 mM AMP-PNP in the absence of ATP, as reported previously for the K978C GOF mutant (panels B, C and Ref. 47). Login to comment
273 The lack of effect on the stability of the activated state is consistent with the virtually complete rescue of the ATP-dependent activity of the E267R bundle mutant that we observed when we combined this mutation with the E1371S catalytic mutation. Login to comment
275 The E267R/E1371S double mutant exhibited high steady-state control currents following activation by ATP and PKA, small relative activation by potentiators and very slow deactivation upon ATP washout similar to that reported for the E1371S single mutant. Login to comment
283 B, macroscopic record showing relatively smaller control current and stronger activation by potentiator addition for a corresponding truncation construct harboring the E267R mutation. Login to comment
290 The E267-K1060 Interaction Also Promotes CFTR Channel Opening in the Absence of ATP Binding or NBD Dimerization- Combining a previously reported GOF mutation (K978C) with the E267R or K1060E bundle mutations revealed that the latter mutations also inhibit ATP-free channel activity both in the presence and absence of NBD2 (i.e. for the èc;1198-CFTR truncation construct). Login to comment
291 The K978C substitution did restore nearly normal ATP-dependent activity of the E267R mutant as evidenced by high control currents in the presence of ATP and PKA and small relative activation by potentiators. Login to comment
294 Conversely, each of the E267R and K1060E mutations substantially reduced the fractional ATP-independent currents exhibited by the full-length K978C-CFTR construct and also reduced the control currents and increased the relative stimulation by potentiators for the NBD2-deletion construct bearing the GOF mutation (K978C/ èc;1198-CFTR). Login to comment
302 The E267R and K1060E mutations in ICL2 and ICL4 both strongly reduced the PKA sensitivity of channel activation in two ways. Login to comment
303 First, in PKA titration experiments the single charge-reversal mutants required much more FIGURE 8. Interplay between the E267R mutation and a site 2 catalytic mutation, E1371S. Login to comment
304 A, macroscopic record showing that the E267R/E1371S double mutant exhibited large control currents, very small stimulation by potentiators and very slow deactivation upon ATP removal. Login to comment
307 B and C, macroscopic records showing that the E267R substitution reduced the baseline current prior to addition of PKA (units/ml) that can be detected for the E1371S-CFTR construct. Login to comment
313 The former interpretation seems likely given that wild type CFTR channels are maximally active at a PKA concentration (200 units/ml) that only partially activated the single E267R and K1060E mutants. Login to comment
321 A-C, macroscopic current records showing that the E267R and K1060E mutants required more PKA than WT-CFTR to achieve only partial activation. Login to comment
325 Symbols are means afe; S.E. Ns are 5, 5, 4, and 5 for WT (black symbols), E267R (red), K1060E (blue), and E267K/K1060E (green), respectively. Login to comment