ABCMdb

PMID: 22612315

Li MS, Cowley EA, Linsdell P
Pseudohalide anions reveal a novel extracellular site for potentiators to increase CFTR function.
Br J Pharmacol. 2012 Nov;167(5):1062-75. doi: 10.1111/j.1476-5381.2012.02041.x., [PubMed]

Sentences

No. Mutations Sentence Comment

36 ABCC7 p.Glu1371Gln Macroscopic current recordings were carried out in an E1371Q-CFTR background (see below). Login to comment 37 ABCC7 p.Arg334Gln ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln In some experiments, additional mutations (R334Q, K892Q, R899Q) were introduced into this background using the QuikChange site-directed mutagenesis system. Login to comment 40 ABCC7 p.Glu1371Gln Patch clamp recording of CFTR In order to monitor and compare CFTR macroscopic conductance in intact cells and in excised membrane patches, we used mutant E1371Q-CFTR channels that show constitutive, high levels of activity when expressed in BHK cells (Zhou et al., 2010; Li et al., 2011). Login to comment 42 ABCC7 p.Glu1371Gln Patch clamp recording of E1371Q-CFTR macroscopic currents was carried out as described in detail recently (Zhou et al., 2010; Li et al., 2011). Login to comment 77 ABCC7 p.Glu1371Gln Experimentally, these regulatory effects of extracellular anions can be monitored and quantified using the constitutively active E1371Q-CFTR mutant, as described in detail previously (Li et al., 2011). Login to comment 78 ABCC7 p.Glu1371Gln With low extracellular Cl- concentrations, E1371Q-CFTR currents in intact cells are small due to block by cytosolic anions, the extent of which can be observed by the increase in current amplitude due to relief of block when the membrane patch is excised to the inside-out configuration (Li et al., 2011). Login to comment 94 ABCC7 p.Glu1371Gln However, with 10 mM Co(NO2)6 3- in the extracellular Figure 1 Effect of external pseudohalide anions on macroscopic E1371Q-CFTR currents in cell-attached and inside-out membrane patches recorded with low extracellular chloride concentration. Login to comment 95 ABCC7 p.Glu1371Gln Example leak-subtracted macroscopic I-V relationships for E1371Q-CFTR recorded under low (4 mM) extracellular Cl-concentration conditions. Constitutively active currents were recorded from cell-attached patches (on cell) and immediately after patch excision (inside out). Login to comment 102 ABCC7 p.Glu1371Gln To gain information on the molecular mechanism of action of extracellular pseudohalide anions, we tested their ability to stimulate CFTR conductance in E1371Q-CFTR channels in which these positively charged residues had been neutralized. Login to comment 104 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln ABCC7 p.Lys892Gln Examples of the macroscopic I-V relationships for R334Q, K892Q, R899Q and K892Q/R899Q (all in an E1371Q background) are shown in Figure 5A. Login to comment 105 ABCC7 p.Arg334Gln Apparent block in intact cells was weak in R334Q and was not significantly weakened further by inclusion of 10 mM Co(CN)6 3- (Figure 5D) or Co(NO2)6 3- (Figure 5E) in the pipette solution. Login to comment 106 ABCC7 p.Arg899Gln Block of R899Q in intact cells was not significantly affected by extracellular Co(CN)6 3- Figure 2 External pseudohalide anions weaken the apparent blocking effect of cytosolic anions under low extracellular chloride concentration conditions. Login to comment 111 ABCC7 p.Lys892Gln Block of K892Q was significantly strengthened by Co(CN)6 3- (Figure 5D) but was unaffected by Co(NO2)6 3- (Figure 5E). Login to comment 112 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Neutralization of both of these extracellular positive charges, in the K892Q/R899Q/ E1371Q triple mutant, resulted in apparent blocker sensitivity that, as in R899Q/E1371Q, was not significantly affected by extracellular Co(CN)6 3- or Co(NO2)6 3- (Figure 5D,E). Login to comment 113 ABCC7 p.Arg334Gln ABCC7 p.Arg899Gln ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln ABCC7 p.Lys892Gln Each of these mutants (R334Q, K892Q, R899Q, K892Q/R899Q) also abolished the sensitivity of current inhibition in intact cells to extracellular Cl- ions (Figure 6). Login to comment 135 ABCC7 p.Glu1371Gln (A) Example leak-subtracted macroscopic I-V relationships for E1371Q-CFTR recorded under high (154 mM) extracellular Cl-concentration conditions. Constitutively active currents were recorded from cell-attached patches (on cell) and immediately after patch excision (inside out). Login to comment 144 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln (A-C) Examples of leak-subtracted macroscopic I-V relationships for different mutants in an E1371Q-CFTR background recorded under low (4 mM) extracellular Cl-concentration conditions (compare E1371Q alone in Figure 1). Login to comment 157 ABCC7 p.Glu1371Gln Evidence that multivalent pseudohalide anions can increase CFTR function comes from the ability of these compounds to increase apparent macroscopic conductance of the constitutively active E1371Q-CFTR mutant in intact cells. Login to comment 158 ABCC7 p.Glu1371Gln Previously, we showed that extracellular Cl- has a similar stimulating effect on E1371Q-CFTR conductance in intact cells, via its ability to destabilize the blockage of open channels by cytosolic substances, blockage which acts to decrease CFTR conductance in a voltage-dependent fashion (Li et al., 2011). Login to comment 162 ABCC7 p.Glu1371Gln Mean fractional current recorded in cell-attached patches relative to inside-out patches for each of the channel variants named (in each case, in an E1371Q background), under low (4 mM) or high (154 mM) extracellular Cl-concentration conditions. Login to comment 170 ABCC7 p.Glu1371Gln Currently, the mechanism by which these compounds stimulate anion secretion in Calu-3 cell monolayers, and its relationship to their effect on E1371Q-CFTR macroscopic conductance in BHK cells, are not known. Login to comment 180 ABCC7 p.Glu1371Gln suggesting they might have effects on these epithelial cells unrelated to their effects on E1371Q-CFTR channels. Login to comment 207 ABCC7 p.Arg334Gln This suggestion of a non-pore-mediated effect is further strengthened by the effects of mutations that remove positive charges from the extracellular face of the CFTR protein. Thus the R334Q mutant, which neutralizes a pore lining positive charge that is known to attract extracellular anions into the outer mouth of the pore (Smith et al., 2001; Gong and Linsdell, 2003c; Zhou et al., 2007), did affect the ability of Co(CN)6 3- and Co(NO2)6 3- to weaken apparent channel block in intact cells (Figures 5, 7). Login to comment 208 ABCC7 p.Arg334Gln However, this apparent lack of pseudohalide sensitivity in R334Q appears to reflect weak cytoplasmic block under control conditions rather than a specific loss of external anion sensitivity of block (Figure 7). Login to comment 209 ABCC7 p.Arg334Gln This interpretation is consistent with the R334Q mutation itself affecting cytoplasmic blocker interactions with CFTR (Zhou et al., 2007). Login to comment 211 ABCC7 p.Arg899Gln Thus, the R899Q mutation showed similar sensitivity to current inhibition in intact cells as wild type, but this inhibition was completely insensitive to extracellular Co(CN)6 3- and Co(NO2)6 3- (Figures 5, 7), suggesting a specific disruption of external anion effect in this mutant. Login to comment 212 ABCC7 p.Lys892Gln Neutralization of another nearby positive charge, in K892Q, had a slightly different effect: block was slightly weakened relative to wild type, was unaffected by Co(NO2)6 3- and was actually significantly strengthened in the presence of external Co(CN)6 3- (Figures 5, 7), again consistent with altered interactions between external anions and cytoplasmic blocking substances in this mutant. Login to comment 214 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln Based on our results with K892Q and R899Q (summarized in Figure 7), we speculate that pseudohalide anions interact with a site away from the channel pore to weaken channel interactions with cytoplasmic blocking substances and so promote elevated overall channel conductance. Login to comment 223 ABCC7 p.Arg899Gln ABCC7 p.Lys892Gln The effects of the K892Q and R899Q mutations point to some involvement of the fourth extracellular loop of CFTR in the regulation of CFTR by extracellular anions. Login to comment