ABCMdb

PMID: 17043152

Aubin CN, Linsdell P
Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.
J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl-concentration. Login to comment 43 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu In contrast to wild-type CFTR, mutants R303E and, to a lesser extent, R352E showed outward rectification of the I-V relationship with symmetrical 154 mM Cl- solutions. Login to comment 63 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu In contrast, both R303E and R352E were associated with strong outward rectification (Fig. 2 A), which is quantified in Fig. 2 B. Login to comment 66 ABCC7 p.Arg352Gln ABCC7 p.Arg303Gln To investigate the role of the charge on these residues in controlling I-V shape, these two arginines were also mutated to neutral glutamine residues; as shown in Fig. 3, R303Q and R352Q were also associated with outward rectification, although in both cases the degree of rectification observed was significantly less than with the charge-changing glutamate substitutions. Login to comment 67 ABCC7 p.Arg303Lys ABCC7 p.Arg352Lys Charge-conservative R303K and R352K mutations were associated with wild type like linear I-V relationships (Fig. 3 A). Login to comment 71 ABCC7 p.Arg303Glu ABCC7 p.Arg303Gln As shown in Fig. 3 B, macroscopic I-V rectification was indeed highly sensitive to symmetrical Cl-concentration in both R303E and R303Q, suggesting that increasing the Cl-concentration partially reverses the effects of removing these positive charges. Login to comment 75 ABCC7 p.Arg352Gln ABCC7 p.Arg303Glu ABCC7 p.Arg303Gln (B) The degree of rectification in R303E, R303Q, and R352Q is dependent on the Cl- concentra- tion. Login to comment 78 ABCC7 p.Arg352Glu Unfortunately, because of low current density in this mutant, R352E could not be studied at low Cl-concentration. Login to comment 95 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu (B) Mean single channel current-voltage relationships constructed from such recordings for wild type (■), R303E (●) and R352E (○). Login to comment 98 ABCC7 p.Arg242Glu ABCC7 p.Arg1102Glu ABCC7 p.Arg933Glu ABCC7 p.Arg80Glu Mutations that did not alter I-V shape (R80E, R242E, R933E, and R1102E) also had no significant effect on unitary conductance (Fig. 4 D). Login to comment 100 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu Single channel currents carried by R303E and R352E under the same symmetrical Cl- solutions used in Fig. 2 are shown in Fig. 5 A. Login to comment 103 ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu ABCC7 p.Arg303Glu For example, at -80 mV, single channel current amplitude was reduced by 89 ± 0% (n = 4) in R303E and by 75 ± 2% (n = 3) in R352E, while at +80 mV, unitary currents were reduced by 30 ± 1% (n = 4) in R303E and by 25 ± 1% (n = 4) in R352E (Fig. 5 B). Login to comment 104 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu The Cl-dependence of I-V shape in R303E and R352E is consistent with removal of positive surface charges that normally act to increase the Cl-concentration at the inner mouth of the pore. Login to comment 112 ABCC7 p.Arg352Cys Unfortunately, R352C Figure 6. Login to comment 114 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu (A) Example single channel currents recorded from wild type, R303E, and R352E at different intracellular Cl- concentrations (154 or 304 mM, as indicated), at a membrane potential of -20 mV. For each trace, the line to the left represents the current level when the channel is closed. Login to comment 119 ABCC7 p.Arg303Cys However, modification of R303C is illustrated in Figs. 7 and 8. Login to comment 124 ABCC7 p.Arg303Cys Similar rundown was also observed in R303C (Fig. 7, A and C; Fig. 8 A), however, the shape of the I-V curve changed during the rundown process, becoming more outwardly rectifying after addition of MTSES (Fig. 7, B and E; Fig. 9) and more inwardly rectifying after application of MTSET (Figs. 8 and 9). Login to comment 125 ABCC7 p.Arg303Gln These changes in I-V shape were not observed in R303Q (unpublished data), suggesting that they are specific for the introduction of a reactive cysteine at this position. Login to comment 126 ABCC7 p.Arg303Glu The results of these experiments are summarized in Fig. 9. Modification of the introduced cysteine residue by negatively charged MTSES leads to pronounced outward rectification, similar to that seen in R303E (Fig. 2 B) and consistent with the deposition of a negative charge at the inner mouth of the pore, while modification by positively charged MTSET resulted in a wild type-like, moderately inwardly rectified I-V relationship. Login to comment 131 ABCC7 p.Arg134Gln ABCC7 p.Arg1030Gln However, both R134Q and R1030Q did yield functional channels (Fig. 10). Login to comment 132 ABCC7 p.Arg134Gln R134Q Figure 7. Modification of CFTR by intracellular MTSES. Login to comment 137 ABCC7 p.Arg303Cys Data from the same patches as in A, recorded before (control) and 6 min after addition of MTSES, are scaled to show the development of outward rectification in R303C but not wild type. Login to comment 142 ABCC7 p.Arg1030Gln R1030Q, like wild type, was associated with an almost linear I-V relationship (Fig. 10, A and B). Login to comment 143 ABCC7 p.Arg134Gln ABCC7 p.Arg134Gln Measurable single channel activity was not observed after R134Q expression; however, Fig. 10 C shows an example of a very small apparent single channel opening frequently observed in a patch excised from an R134Q-expressing cell. Login to comment 144 ABCC7 p.Arg134Gln This tiny current suggests that R134Q has an extremely small unitary conductance below the accurate resolution of single channel recording. Login to comment 145 ABCC7 p.Arg1030Gln R1030Q single channel currents were readily observed (Fig. 10 C), and these channels showed a slightly, but significantly, reduced single channel conductance (Fig. 10 D) of 4.70 ± 0.04 pS (n = 4) compared with 5.39 ± 0.06 pS (n = 5) for wild type (P < 0.0001, two-tailed t test). Login to comment 149 ABCC7 p.Arg242Glu ABCC7 p.Arg352Gln ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu ABCC7 p.Arg1102Glu ABCC7 p.Arg933Glu ABCC7 p.Arg80Glu Under these conditions, current carried not only by wild-type CFTR, but also by each of the channel mutants R303E, R352E (Fig. 10), R80E, R242E, R933E, R1102E, and R352Q (not depicted) showed reversal potentials that were not significantly different from the calculated Cl- equilibrium potential (+33.4 mV). Login to comment 152 ABCC7 p.Arg303Cys (A) Individual examples of the time course of MTSET-induced current rundown in wild type and R303C. Login to comment 172 ABCC7 p.Arg134Gln ABCC7 p.Arg1030Gln (A) Example leak-subtracted macroscopic I-V relationships from R134Q and R1030Q-CFTR in inside-out membrane patches with symmetrical 154 mM Cl- solu- tions, after maximal channel activation. Login to comment 174 ABCC7 p.Arg134Gln * indicates a significant difference from wild type at the same Cl-concentration (P < 10-4, two-tailed t test), † indicates a significant difference from R134Q at 154 mM Cl- (P < 0.005, two-tailed t test). Login to comment 177 ABCC7 p.Arg134Gln For R134Q, the tiny current associated with a putative channel opening is identified by a line above it. Login to comment 179 ABCC7 p.Arg1030Gln (D) Mean single channel current-voltage relationships for wild type (filled circles) and R1030Q (open circles). Login to comment 181 ABCC7 p.Arg352Cys to poor expression of the R352C mutant, and as a result it remains uncertain whether this charged amino acid contributes directly to pore surface charge. Login to comment 184 ABCC7 p.Arg352Gln ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu ABCC7 p.Arg303Gln Furthermore, rectification in R303Q and R303E appeared more sensitive to the intracellular Cl-concentration than in R352Q and R352E (Fig. 3 B). Login to comment 196 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu Unfortunately we were unable to obtain single channel recordings at Cl- concentrations higher than 304 mM; however, the dramatic increase in conductance seen in both R303E and R352E when intracellular [Cl-] was increased from 154 to 304 mM (Fig. 6) suggests that the conductance of these mutant channels is far from saturated at this elevated Cl-concentration, consistent with a large increase in Km. Login to comment 199 ABCC7 p.Arg352Glu ABCC7 p.Arg303Glu Nevertheless, unitary currents carried by Cl- influx were also reduced in both R303E and R352E (Fig. 5). Login to comment 206 ABCC7 p.Arg242Glu ABCC7 p.Arg352Gln ABCC7 p.Arg1102Glu ABCC7 p.Arg933Glu ABCC7 p.Arg80Glu Examples of similar data obtained from three to four patches for these channel variants and also for R80E, R242E, R352Q, R933E, and R1102E (not depicted). Login to comment 207 ABCC7 p.Arg134Gln ABCC7 p.Arg1030Gln R134Q and R1030Q do not appear consistent with loss of an intracellular surface charge. Login to comment 208 ABCC7 p.Arg352Gln ABCC7 p.Arg303Glu ABCC7 p.Arg303Gln ABCC7 p.Arg134Gln ABCC7 p.Arg134Gln R134Q is associated with outward rectification (Fig. 10, A and B); however, this effect shows a complex Cl-dependence that, unlike that of R303E, R303Q, and R352Q (Fig. 3 B), is not consistent with a simple surface charge effect. Furthermore, R134Q shows an extremely small apparent unitary conductance (Fig. 10 C). Login to comment 210 ABCC7 p.Arg1030Gln R1030Q did not alter macroscopic current rectification (Fig. 10 B) but was associated with a small decrease in unitary conductance (Fig. 10 D), suggesting that while this residue may play a role in determining pore properties, it does not act as an important inner surface charge. Login to comment 211 ABCC7 p.Arg1030Glu Unfortunately we were unable to identify currents associated with R1030E, although this mutant was previously expressed in another mammalian cell line (Anderson et al., 1991); this previous study did not note any changes in pore properties associated with this mutant. Login to comment