ABCMdb

PMID: 23478129

Billet A, Mornon JP, Jollivet M, Lehn P, Callebaut I, Becq F
CFTR: effect of ICL2 and ICL4 amino acids in close spatial proximity on the current properties of the channel.
J Cyst Fibros. 2013 Dec;12(6):737-45. doi: 10.1016/j.jcf.2013.02.002. Epub 2013 Mar 9., [PubMed]

Sentences

No. Mutations Sentence Comment

72 ABCC7 p.Glu267Ala ABCC7 p.Lys1060Ala ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu 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 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu We also studied the double mutant E267R-K1060E-CFTR, where the negative and positive charges are inverted. Login to comment 86 ABCC7 p.Glu267Ala ABCC7 p.Glu267Ala ABCC7 p.Glu267Ala ABCC7 p.Lys1060Ala ABCC7 p.Lys1060Ala ABCC7 p.Lys1060Ala ABCC7 p.Glu267Arg ABCC7 p.Glu267Arg ABCC7 p.Glu267Arg ABCC7 p.Glu267Arg ABCC7 p.Glu267Arg ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu ABCC7 p.Lys1060Glu ABCC7 p.Lys1060Glu ABCC7 p.Lys1060Glu ABCC7 p.Lys1060Glu ABCC7 p.Lys1060Glu 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 ABCC7 p.Glu267Ala ABCC7 p.Lys1060Ala ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu 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 105 ABCC7 p.Glu267Ala First, the presence of a small and uncharged residue at the E267 position (E267A mutant) decreased by approximately 50% the Cl-current density compared to wt. Login to comment 106 ABCC7 p.Glu267Arg 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 107 ABCC7 p.Glu267Ala ABCC7 p.Lys1060Ala ABCC7 p.Lys1060Glu 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 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu Finally, with the "inverted" double mutant (E267R-K1060E) the recorded Cl-current density was not significantly different from wt CFTR. Login to comment 113 ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Ser263His ABCC7 p.Ser263His To investigate the influence of these two amino acids on CFTR channel function, we substituted one or both residues by a histidine residue, which is a potentially positively charged residue as it is protonable in a pH-dependent manner (pKa of ~6), creating thereby the mutants S263H- and V1056H- as well as the double-mutant S263H-V1056H-CFTR. Login to comment 116 ABCC7 p.Val1056His ABCC7 p.Ser263His Importantly upon addition of forskolin, the electrophysiological properties of the ICL2 S263H and ICL4 V1056H mutants were clearly different from those observed with wt CFTR: the CFTR current observed with both mutants was time and voltage dependent (Fig. 3C-D). Login to comment 117 ABCC7 p.Val1056His ABCC7 p.Ser263His At positive potentials, the outward current of S263H and V1056H mutants greatly increases compared to wt, but contrary to wt CFTR currents, it deactivates during the pulse potential (Figs. 3C-D, 4). Login to comment 120 ABCC7 p.Val1056His ABCC7 p.Ser263His Interestingly, in the case of the double mutant S263H-V1056H, we observed the restoration of the time and voltage independence of the current as well as of the level of the current density (Fig. 3C-D, Table 1). Login to comment 137 ABCC7 p.Glu267Ala ABCC7 p.Lys1060Ala We observed that the abolition of one charged residue in the E267A or K1060A mutants induced a 50% diminution of the Cl-current density (Fig. 2). Login to comment 140 ABCC7 p.Glu267Arg 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 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu 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 143 ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Ser263His ABCC7 p.Ser263His ABCC7 p.Ser263His ABCC7 p.Ser263His -100 -50 100 50 C D A S263H-V1056H S263H V1056H I (pA /pF) V (mV) S263H V1056H S263H-V1056H 5000pA 50ms B 600 400 200 -200 Fig. 3. Login to comment 150 ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Ser263His ABCC7 p.Ser263His (C, D) Whole cell chloride current of HEK293 cells expressing S263H-CFTR, V1056H-CFTR or the double mutant S263H-V1056H-CFTR obtained in presence of 10 bc;M Fsk. Login to comment 174 ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Ser263His ABCC7 p.Ser263His ABCC7 p.Ser263His ABCC7 p.Ser263His I (pA/pF) at -100 mV Basal Fsk/start of the pulse Fsk/end of the pulse +CFTRinh172 Mean SEM N Mean SEM N t test Mean SEM N t test Mean SEM N wt -3.4 0.9 18 -100.4 17.5 18 -74.3 7.3 18 -11.2 3.0 18 S263H -3.1 0.8 8 -207.0 37.8 8 ** -84.6 13.9 8 ns -5.9 1.0 8 V1056H -2.0 0.6 7 -213.0 31.6 7 ** -95.0 18.7 7 ns -4.1 0.9 7 S263H-V1056H -1.8 0.4 16 -96.0 14.5 16 ns -80.5 9.3 16 ns -7.9 1.0 16 I (pA/pF) at +100 mV Basal Fsk/start of the pulse Fsk/end of the pulse +CFTRinh172 Mean SEM N Mean SEM N t test Mean SEM N t test Mean SEM N wt 14.5 4.1 18 260.5 46.1 18 233.6 25.0 18 35.9 6.2 18 S263H 13.1 3.5 8 573.5 75.5 8 ** 523.7 60.5 8 *** 25.8 6.7 8 V1056H 5.7 1.9 7 588.8 68.6 7 *** 531.9 65.6 7 *** 14.9 4.2 7 S263H-V1056H 7.8 1.2 16 279.1 40.5 16 ns 264.7 30.7 16 ns 14.0 4.8 16 ns: not significant difference; **: pb0.01; ***: pb0.001. Login to comment 177 ABCC7 p.Val1056His ABCC7 p.Ser263His Clearly, the understanding of the reasons underlying the outward rectification seen in both S263H and V1056H mutants requires further in-depth investigations, nevertheless the effects of those mutations on current density and voltage-dependence suggest that this region of the protein may play a critical role in the Cl- pathway and may be physically close to the inner mouth of the pore. Login to comment 178 ABCC7 p.Val1056His ABCC7 p.Val1056His ABCC7 p.Ser263His ABCC7 p.Ser263His Restoration of the voltage independence and of a current density similar to wt in the double mutant S263H-V1056H might be explained by the probable tight contact between the two histidine residues, similarly to the situation occurring in the Sav1866 template, a contact possibly counteracting the effects of the single mutant (S263H or V1056H) on current density. Login to comment