ABCMdb

PMID: 18449561

Zhou JJ, Fatehi M, Linsdell P
Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.
Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. Login to comment 60 ABCC7 p.Lys335Glu ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu ABCC7 p.Arg1128Glu As shown in Fig. 2, similar inward rectification is observed in the charge-reversing mutants R104E, R117E, and K335E and to a much lesser extent, R1128E, under symmetrical ionic conditions in excised membrane patches. Login to comment 61 ABCC7 p.Lys892Glu ABCC7 p.Arg899Glu ABCC7 p.Lys329Glu ABCC7 p.Lys114Glu In contrast, K114E, K329E, K892E, and R899E, like wild type, gave almost linear I-V relationships under these conditions. Login to comment 62 ABCC7 p.Lys335Ala ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg1128Gln To investigate the role of charge on these residues in controlling I-V shape, neutral substitutions (R104Q, R117Q, K335A, R1128Q) were also investigated. Login to comment 67 ABCC7 p.Lys335Glu ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu In contrast to the linear I-V relationship seen in wild-type CFTR with symmetrical 154 mM Cl- solutions, mutants R104E, R117E, and K335E showed clear inward rectification. Login to comment 73 ABCC7 p.Lys335Glu ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu As shown in Fig. 3b, macroscopic current rectification was indeed sensitive to symmetrical Cl-concentration in R104E, R117E, and K335E, being more pronounced at low Cl-concentration. Login to comment 78 ABCC7 p.Arg1128Glu ABCC7 p.Lys892Glu ABCC7 p.Arg899Glu ABCC7 p.Lys329Glu ABCC7 p.Lys114Glu The mutations K114E, K329E, K892E, R899E, and R1128E did not affect unitary current amplitude at any voltage (Fig. 4b), consistent with these residues not being involved in Cl- permeation. Login to comment 79 ABCC7 p.Lys335Glu ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu However, current amplitude was significantly reduced in R104E, R117E, and K335E (Fig. 4a, b). Login to comment 89 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys The expected side chain charge at R104C and R117C following modification by these reagents mirrored the effects of mutation to Fig. 3 Charge and chloride dependence of current rectification in mutant forms of CFTR. Login to comment 91 ABCC7 p.Lys335Glu ABCC7 p.Lys335Ala ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu ABCC7 p.Arg1128Glu ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg1128Gln All mutants depicted (R104Q, R117Q, K335A, R1128Q, R104E, R117E, K335E, R1128E) showed rectification ratios significantly different from wild type (asterisk P<0.05). Login to comment 92 ABCC7 p.Lys335Glu ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu b The degree of rectification in R104E, R117E, and K335E is dependent on the Cl-concentration. Login to comment 95 ABCC7 p.Arg117Cys ABCC7 p.Arg117Glu Mean of data from four to six patches in both a and b side chains bearing different charges (Fig. 5b), with the possible exception that R117C modified by MTSES did not show the same degree of inward rectification seen in R117E. Login to comment 100 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys However, using the same MTS pretreatment protocols as in our previous study [2] (see "Materials and methods"), we found that both R104C and R117C could be modified by both MTSET and MTSES prior to channel activation, effectively mimicking the effects of inclusion of these substances in the pipette solution on rectification of the I-V relationship (Fig. 6). Login to comment 101 ABCC7 p.Arg117Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Arg104Cys This suggests that both positively and negatively charged MTS reagents can modify both R104C and R117C independently of the state of channel activation, a situation that contrasts with R334C, K335C, and other TM6 mutants. Login to comment 103 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys As shown in Fig. 7, external application of pCMBS also increased the inward rectification seen in R104C and R117C, consistent with deposition of a negative charge on the cysteine present at these positions. Login to comment 104 ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu In fact, at both sites, pCMBS had a greater effect on the rectification ratio than MTSES (P<0.05), making them closer to results obtained with R104E and R117E. Login to comment 116 ABCC7 p.Lys335Glu ABCC7 p.Lys335Ala ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln All mutants depicted (R104Q, R117Q, K335A, R104E, R117E, K335E) significantly different from wild type (asterisk P<0.05). Login to comment 120 ABCC7 p.Lys335Ala ABCC7 p.Arg104Gln ABCC7 p.Arg1128Gln As shown in Fig. 8a, while Pt(NO2)4 2-induced strong inward rectification in wild type (as a result of strongly voltage-dependent current inhibition) [26], this effect was less marked in R104Q, K335A, and R1128Q. Login to comment 122 ABCC7 p.Lys335Ala ABCC7 p.Lys329Ala ABCC7 p.Arg117Gln ABCC7 p.Arg104Gln ABCC7 p.Arg1128Gln ABCC7 p.Arg899Gln ABCC7 p.Lys114Cys Considering the data at +80 mV, where the inhibitory effects of Pt(NO2)4 2- are strongest, suggests that Pt(NO2)4 2- inhibition is significantly weakened in R104Q, K335A, and (to a lesser extent) R1128Q but not significantly altered in K114C, R117Q, K329A, K829Q, or R899Q (Fig. 8c). Login to comment 125 ABCC7 p.Arg117Gln ABCC7 p.Arg1128Gln Similar effects were observed in R117Q and R1128Q (Fig. 9b), consistent with the minor effects of these mutations on Pt(NO2)4 2- inhibition inferred from the rectification of macroscopic currents (Fig. 8). Login to comment 126 ABCC7 p.Lys335Ala ABCC7 p.Arg104Gln In contrast, unitary current amplitude in both R104Q and K335A appeared relatively resistant to the inhibitory effects of external Pt(NO2)4 2- ions (Fig. 9a-c), again consistent with effects on macroscopic currents (Fig. 8), suggesting that positive charge at positions 104 and 335 is necessary for the full inhibitory effects of extracellular Pt(NO2)4 2- ions. Login to comment 128 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys Our present study, together with previous work on R334 [6,8,22,26], has surveyed the effects of removing all permanent positive charges (contributed by arginine and lysine side chains) in the outer TMs and ECLs on the permeation properties of Fig. 6 Modification of R104C and R117C by MTSES is independent of channel activation. Login to comment 131 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys b Mean rectification ratios for R104C (left) and R117C (right) under control conditions (open bars) and following modification by MTSET or MTSES using a pretreatment protocol (black bars) or by inclusion in the pipette (gray bars; see Fig. 5). Login to comment 134 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys a Example relative current-voltage (IREL-V) relationships for R104C (left) and R117C (right) under control conditions and with MTSET or MTSES present in the pipette solution. Login to comment 137 ABCC7 p.Arg117Glu The charge on the side chain is assumed to be +1 (MTSET, WT), 0 (R104/ 117C, R104/117Q), or -1 (MTSES, R104/R117E). Login to comment 144 ABCC7 p.Arg117His Slightly reduced unitary conductance has previously been reported in the cystic fibrosis-associated mutant R117H [9, 20]; we suggest that this effect results from partial removal of this important positive charge and its electrostatic attractive effects on external Cl- ions. Login to comment 145 ABCC7 p.Lys335Glu ABCC7 p.Lys335Glu ABCC7 p.Arg104Glu ABCC7 p.Arg104Glu ABCC7 p.Arg104Gln ABCC7 p.Arg104Gln The reduction in unitary current for Cl-efflux seen in R104Q, R104E, and K335E (Fig. 4) further suggests that these residues may play an electrostatic role in Fig. 8 Mutation of positively charged residues weakens the apparent inhibitory effect of external Pt(NO2)4 2- ions. Login to comment 147 ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Arg104Gln ABCC7 p.Arg104Gln ABCC7 p.Arg1128Gln ABCC7 p.Arg1128Gln b Mean fraction of control normalized current remaining in the presence of 10 mM extracellular Pt(NO2)4 2at different membrane potentials in wild type (closed circles), R104Q (closed squares), K335A (open circles), and R1128Q (open squares), quantified as the IREL in the presence of 10 mM Pt(NO2)4 2- [IREL(block)] as a fraction of that in the absence of Pt(NO2)4 2- [IREL(control)]. Login to comment 150 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys Mean of data from four to six patches in both b and c Fig. 7 Modification of R104C and R117C by pCMBS. Login to comment 152 ABCC7 p.Arg117Cys ABCC7 p.Arg104Cys b Mean rectification ratios for R104C (left) and R117C (right) under control conditions (open bars) and following modification by pCMBS using a pretreatment protocol (black bars) or by inclusion in the pipette (gray bars). Login to comment 162 ABCC7 p.Arg104Gln ABCC7 p.Lys335Gln These inhibitory effects are weakened in R104Q and K335Q (Figs. 8 and 9), consistent with the idea that Pt(NO2)4 2- ions may interact with these positive charges to interfere with Cl- permeation. Login to comment 170 ABCC7 p.Lys335Ala ABCC7 p.Arg104Gln c Mean fractional unitary current amplitude remaining in the presence of this concentration of Pt (NO2)4 2- ions as a function of voltage, in wild type (open circles), R104Q (closed circles, left panel), and K335A (closed circles, right panel). Login to comment 172 ABCC7 p.Arg1128Gln In contrast, data for R107Q and R1128Q were not significantly different from wild type at any voltage (P>0.05). Login to comment