ABCMdb

ABCC7 p.Lys1060Glu

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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
107 For K1060, irrespective of the replacement of the positively charged side chain by a small uncharged residue (K1060A mutant) or a negatively charged residue (K1060E mutant), the inward and outward Cl-current densities were 50-60% lower than the corresponding Cl-currents elicited by wt channels but similar to that of the E267A mutant. 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
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
138 Fig. 5, A--C confirm this prediction for an E267K/K1060E double mutant. 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
157 D, K1060E-CFTR channels also exhibit reduced control currents and strong activation by potentiators. 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
177 Conversely introducing a charge swap double mutation (E267K/K1060E) had no apparent effect on the basal currents and potentiator responses of this truncation construct (Fig. 7, C-E). 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
187 The magnitudes of the unitary currents at this holding potential were not different from those for WT-CFTR as follows: WT, 0.427 afe; 0.003 pA (mean afe; S.E.) measured for 206 openingsin2patches;E267R,0.419afe;0.005pAfor119openingsin3patches; K1060E, 0.431 afe; 0.004 pA for 166 openings in 3 patches. Login to comment
189 The mean open channel burst durations calculated by cycle time analysis (see"ExperimentalProcedures")were0.7afe;0.1and0.6afe;0.1sforE267R-CFTR (n afd; 6 patches) and for K1060E-CFTR (n afd; 4), respectively. 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
205 Importantly, normal PKA sensitivity was restored by introducing charge swap mutations across the putative interface (E267K/K1060E; Fig. 9, D-F). Login to comment
241 A, macroscopic current record for a double mutant with charge-reversal substitutions at both positions (E267K/K1060E-CFTR). Login to comment
248 See also Fig. 2E for K1060E single mutant data. Login to comment
252 Error bars (S.E.) for WT and E267/K1060E are too small to be seen. 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
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
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
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
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