ABCMdb

PMID: 25892339

Linsdell P
Interactions between permeant and blocking anions inside the CFTR chloride channel pore.
Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Glu1371Gln In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. Login to comment 3 ABCC7 p.Ile344Lys A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4 2-binding affinity also greatly strengthened antagonistic Cl- :blocker interactions as well as the voltage-dependence of block. Login to comment 4 ABCC7 p.Ile344Lys Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4 2- ions but also with extracellular Cl- , and that altered blocker Cl- - and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl-binding in the outer vestibule. Login to comment 6 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys However, the I344K mutation does not increase single channel conductance following disruption of Cl-binding in the outer vestibule in R334Q channels. Login to comment 32 ABCC7 p.Glu1371Gln Binding of Pt(NO2)4 2-to this site in the inner vestibule affects both Cl-permeation through the open channel and also channel opening and closing [47], however the effect on Cl- movement in open channels can be isolated using gating-defective, constitutively open E1371Q-CFTR channels [47]. Login to comment 40 ABCC7 p.Glu1371Gln Macroscopic currents were recorded from gating-defective, constitutively-open E1371Q channels [49], whereas single channel recording experiments were carried out in a wild type CFTR background. Login to comment 41 ABCC7 p.Glu1371Gln It has previously been shown that, whereas Pt(NO2)4 2- has complex effects on Cl- conduction and gating in wild type, effects on conduction are isolated in E1371Q, and that blocking effects on this channel mutant are indistinguishable from those on wild type channels in the open state [47]. Login to comment 65 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln (A, B, E, F) Example macroscopic I-V relationships for E1371Q (A, B) or R899Q/E1371Q (E, F) under high (154 mM; A, E) or low (4 mM; B, F) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of 100 bc;M and 1 mM Pt(NO2)4 2-to the intracellular solution. Login to comment 66 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln (C, G) Mean fraction of control current remaining after addition of different concentrations of Pt(NO2)4 2- at a membrane potential of -100 mV for E1371Q (C) and R899Q/E1371Q (G) under high (filled circles) and low (open circles) extracellular [Cl- ] conditions. Data have been fitted using Eq. (1) as described in the Materials and methods. Login to comment 67 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln (D) Mean Pt KD values obtained from such fits at different membrane potentials for E1371Q; results for R899Q/E1371Q were indistinguishable (not shown). Login to comment 69 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln (H) Mean values of Pt KD at 0 mV membrane potential obtained from fits of Pt KD-V relationships such as those shown in D for E1371Q (filled squares) and R899Q/E1371Q (open squares) at different extracellular [Cl- ]. Login to comment 71 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln There was no significant difference between E1371Q and R899Q/E1371Q (P N 0.85). Login to comment 73 ABCC7 p.Glu1371Gln dissociation constant for block by intracellular Pt(NO2)4 2- ions (Pt KD) at different extracellular [Cl- ] ([Cl- ]o) derives from both the dissociation constant for the internal binding site when the external binding site is occupied by Cl- (Pt Kocc) and that when the external binding site is vacant (Pt Kvac) according to: Pt KD &#bc; Pt Kocc Cl - &#bd; o &#fe; Pt Kvac: &#f0;3&#de; This model predicts a linear relationship between Pt KD and [Cl- ]o, which was reasonably well observed in the present study at different membrane potentials for E1371Q (Figs. 2H, 8A) and pore-mutant CFTR channels (Figs. 4E, J, 6D, H, 8B, C, 10A, B), as well as when extracellular Cl-was replaced by other anions (Figs. 3D, 7E, F, 11A, E). Login to comment 82 ABCC7 p.Glu1371Gln This effect is illustrated for constitutively open E1371Q channels in Fig. 2: Pt(NO2)4 2- block is strengthened as extracellular Cl- is replaced by impermeant gluconate anions (Fig. 2C, D), resulting in a monotonic increase in the measured KD (at 0 mV membrane potential) as extracellular [Cl- ] is increased (Fig. 2H). Login to comment 83 ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln Identical results were obtained in R899Q/ E1371Q channels (Fig. 2; Table 1), both at high and low [Cl- ]o conditions, indicating that extracellular Cl- interactions with this externally-located arginine residue (Fig. 1) do not contribute to these antagonistic effects. Login to comment 84 ABCC7 p.Glu1371Gln This confirms that use of the E1371Q background effectively isolates Cl- :Pt(NO2)4 2- interactions that occur within the channel pore from those that may involve Cl- interactions with a non-pore extracellular site. Login to comment 87 ABCC7 p.Glu1371Gln (A, B) Example macroscopic I-V relationships for E1371Q when the extracellular solution contained 150 mM SCN- (A) or 150 mM ClO4 - (B). Login to comment 99 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Thus, K95Q/E1371Q is associated with weakened Pt(NO2)4 2- block (Fig. 4A-E), and I344K/E1371Q with greatly strengthened block (Fig. 4F-J). Login to comment 101 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Thus Pt KD was only very weakly [Cl- ]o-sensitive in K95Q/E1371Q (Fig. 4E), but very strongly [Cl- ]o-dependent in I344K/E1371Q (Fig. 4J). Login to comment 102 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys The affinity, voltage dependence, and [Cl- ]o-dependence of block in E1371Q, K95Q/E1371Q and I344K/E1371Q are compared directly in Fig. 5 and Table 1. Login to comment 105 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln Consistent with this model, neutralization of this charge in the R334Q/E1371Q mutant abolished the relationship between intracellular Pt(NO2)4 2- block and extracellular [Cl- ] (Fig. 6A-D). Login to comment 106 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Block under low [Cl- ] conditions was weakened by the R334Q mutation (Table 1; also compare results for R334Q/E1371Q in Fig. 6C with E1371Q in Fig. 2D), resulting in a significant increase in Pt KD at all voltages (P b 0.002). Login to comment 108 ABCC7 p.Arg334Gln The R334Q mutation also appeared to increase the voltage-dependence of block (Table 1), although the reasons for this effect are not clear. Login to comment 109 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Since the R334Q and I344K mutations in different parts of the pore (Fig. 1) had opposite effects on the [Cl- ]o-sensitivity of Pt(NO2)4 2- block - which was practically abolished in R334Q/E1371Q (Fig. 6) but greatly increased in I344K/E1371Q (Figs. 4, 5) - these mutations were combined to generate a R334Q/I344K/E1371Q mutant. Login to comment 110 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Not only did R334Q/I344K/E1371Q channels exhibit strong Pt(NO2)4 2- block (Table 1), but block was also strongly dependent on extracellular [Cl- ] (Fig. 6E-H). Login to comment 111 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys This confirms that the I344K mutation not only strengthens interactions with intracellular Pt(NO2)4 2- ions but also with extracellular Cl-ions. Furthermore, this effect on interactions with extracellular Cl- appears independent of the presence of a positively charged side chain at position 334: whereas the R334Q mutation abolishes Cl- -dependence of block in E1371Q channels (Fig. 6D), it does not in I344K- bearing E1371Q channels (Fig. 6H). Login to comment 112 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Thus, interactions with external Cl- that are lost in R334Q/E1371Q channels are at least partially restored by the I344K mutation. Login to comment 113 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys To examine if the I344K mutation also resulted in altered interactions with other extracellular anions - and if this effect was also independent of R334 -Pt(NO2)4 2- block was also investigated with other extracellular anions, both in I344K/E1371Q and in R334Q/I344K/ E1371Q channels (Fig. 7). Login to comment 114 ABCC7 p.Glu1371Gln In both these channel mutants Pt(NO2)4 2- block was also weakened by other extracellular anions (Fig. 7C-H), with a similar overall apparent anion selectivity to that observed for E1371Q (Fig. 3E). Login to comment 116 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys First, SCN- had a very strong negative effect on Pt(NO2)4 2- block in I344K/E1371Q, increasing Pt KD(0) N600-fold relative to that with gluconate and N25-fold relative to that with Cl- (Fig. 7C, E, G). Login to comment 117 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Secondly, formate was able to significantly increase Pt KD(0) relative to gluconate in both I344K/E1371Q (Fig. 7G) and in R334Q/I344K/E1371Q (Fig. 7H), unlike in E1371Q where formate had no significant effect (Fig. 3D, E). Login to comment 119 ABCC7 p.Glu1371Gln A quantitative model of anion binding In constitutively open E1371Q channels, extracellular Cl- (Fig. 2) and other monovalent anions (Fig. 3) weaken the blocking effects of Pt(NO2)4 2- , suggesting that these anions compete for binding inside the channel pore. Login to comment 123 ABCC7 p.Glu1371Gln Fits to mean data for E1371Q using Eq. (3) (Fig. 8A) suggest, at 0 mV membrane potential, a Pt Kvac of 245 bc;M (Fig. 8D), Pt Kocc of 1440 bc;M (Fig. 8E), and Cl K 179 mM (Fig. 8F), with voltage dependencies of these parameters suggesting Pt zb4;vac of -0.39 (Fig. 8D), Pt zb4;occ -0.63 (Fig. 8E), and Cl zb4; +0.22 (Fig. 8F). Login to comment 126 ABCC7 p.Glu1371Gln Asterisks indicate a significant difference from E1371Q (*P b 0.05; **P b 0.001). Login to comment 128 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Pt KD(0) (4 mM Cl- ) (bc;M) zb4; (4 mM Cl- ) Pt KD(0) (154 mM Cl- ) (bc;M) zb4; (154 mM Cl- ) E1371Q 183.7 &#b1; 33.2 (9) -0.397 &#b1; 0.030 (9) 441.0 &#b1; 28.6 (7) -0.503 &#b1; 0.026 (7) R899Q/E1371Q 189.9 &#b1; 55.0 (6) -0.362 &#b1; 0.063 (6) 434.0 &#b1; 32.2 (7) -0.458 &#b1; 0.048 (7) K95Q/E1371Q 1110 &#b1; 172 (6)** -0.244 &#b1; 0.022 (6)* 1422 &#b1; 218 (6)** -0.193 &#b1; 0.041 (6)** I344K/E1371Q 6.92 &#b1; 1.48 (6)** -1.589 &#b1; 0.125 (6)** 164.7 &#b1; 27.5 (7)** -1.604 &#b1; 0.080 (7)** R334Q/E1371Q 1081 &#b1; 220 (4)** -0.637 &#b1; 0.106 (4)* 1112 &#b1; 144 (4)** -0.621 &#b1; 0.051 (4)* R334Q/I344K/E1371Q 39.24 &#b1; 7.94 (4)* -1.093 &#b1; 0.037 (4)** 258.3 &#b1; 30.7 (5)* -1.075 &#b1; 0.033 (5)** Fig. 4. Effect of mutations that weaken or strengthen intracellular Pt(NO2)4 2- block. Login to comment 129 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys (A, B, F, G) Example macroscopic I-V relationships for K95Q/E1371Q (A, B) or I344K/E1371Q (F, G) under high (154 mM; A, F) or low (4 mM; B, G) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of Pt(NO2)4 2- (at the concentrations indicated) to the intracellular solution. Login to comment 135 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln There was no significant difference in these parameters for K95Q/E1371Q (E) (P N 0.05). Login to comment 136 ABCC7 p.Glu1371Gln Dotted line indicates the fit to data for E1371Q (see Fig. 2H). Login to comment 140 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Quantitative analysis of anion binding in mutant channels Similar analysis of Cl- -dependent Pt(NO2)4 2- block in K95Q/E1371Q and I344K/E1371Q (Fig. 8B, C) provides additional insight into the effect of mutations close to the putative Pt(NO2)4 2-binding site. Login to comment 142 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Compared to E1371Q, Pt(NO2)4 2-binding was weakened in K95Q/E1371Q, both in Cl- - unoccupied (Fig. 8D) and Cl- -occupied channels (Fig. 8E), although its voltage dependence was little changed; and Cl-binding itself was only slightly weakened (Fig. 8F). Login to comment 143 ABCC7 p.Lys95Gln Since K95 is thought to play a dominant role in Cl-binding in the inner vestibule [34,51,52], the finding that the K95Q mutation has only a small effect on Cl-binding affinity is consistent with the hypothesis that external Cl- is binding to a different site located in a more extracellular part of the channel. Login to comment 144 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln In fact, since Pt(NO2)4 2- block is so weak in K95Q/E1371Q, the accuracy of estimated Cl K in this mutant is questionable. Login to comment 145 ABCC7 p.Ile344Lys The I344K mutant has a profound impact on channel binding affinity of both intracellular Pt(NO2)4 2- (Fig. 8D, E) and extracellular Cl- (Fig. 8F) (summarized in Table 2). Login to comment 146 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Compared to E1371Q, the intrinsic Pt(NO2)4 2-binding affinity of I344K/E1371Q is increased ~1400-fold by the addition of a second fixed positive charge in the inner vestibule, with no apparent change in the voltage dependence of binding (Fig. 8D). Login to comment 148 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys In fact, the impact of Cl-binding on Pt(NO2)4 2-binding affinity (the difference between Pt Kvac and Pt Kocc) appears much greater in I344K/E1371Q than in E1371Q (Fig. 9). Login to comment 149 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Finally, Cl-binding affinity is higher in I334K/E1371Q (Fig. 8F; Table 2), suggesting that the I344K mutation strengthens interactions with external Cl-ions as well as with internal Pt(NO2)4 2- ions. Furthermore, the voltage-dependence of Cl-binding is greatly increased, with Cl zb4; being increased N5-fold (Fig. 8F), suggesting that external Cl-ions cross a much larger part of the transmembrane electric field to reach their binding site in this mutant. Login to comment 150 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln External Cl- -dependence of Pt(NO2)4 2- block is abolished in R334Q/ E1371Q (Fig. 6C, D; Fig. 10A), consistent with the positive charge at this site in the outer vestibule of the pore (Fig. 1) being crucial for interactions between the channel and extracellular Cl-ions that are required for ion:ion interactions inside the pore [33,43]. Login to comment 151 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Binding of Pt(NO2)4 2- ions to R334Q/E1371Q was somewhat weaker than E1371Q (Fig. 10C; Table 2), consistent with earlier reports that mutations at R334 disrupt blocker binding in the pore inner vestibule, perhaps by a long-range conformational effect on the pore [56]. Login to comment 152 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln The voltage-dependence of Pt(NO2)4 2-binding is also increased in R334Q/E1371Q (Fig. 10C). Login to comment 153 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln However, the very low apparent Cl- affinity of R334Q/E1371Q implied by the apparent [Cl- ]-independence of Pt KD in this mutant (Figs. 6D, 10A) precluded quantitative analysis of Pt Kocc(0) and Cl K in this case; the lack of slope illustrated in the relationships in Figs. 6D and 10A imply extremely low Cl-binding affinity, consistent with ablation of the external Cl-binding site. Login to comment 154 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Although the R334Q mutant abolished Cl- -dependence of Pt(NO2)4 2- block, the double pore mutant R334Q/I344K/E1371Q showed strongly Cl- -dependent block (Fig. 6E-H; Fig. 10B), suggesting that the presence of the I344K mutant restores Cl-binding that is lost in R334Q/E1371Q. Login to comment 155 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Quantitative analysis of data from R334Q/I344K/ E1371Q (Fig. 10; Table 2) suggests that the presence of the second fixed positive charge in the inner vestibule at position 344 still supports strong Pt(NO2)4 2-binding (Fig. 10C), although (as for E1371Q) Pt(NO2)4 2-binding is somewhat weakened by the R334Q mutation (Fig. 10C). Login to comment 156 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Binding of Pt(NO2)4 2-to Cl- -occupied channels is similar in R334Q/E1371Q and R334Q/I344K/E1371Q (Fig. 10D), suggesting that R334 plays little or no role in ion:ion interactions in mutant channels bearing a positive charge at position 344. Login to comment 157 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Chloride binding also remains strong in R334Q/I344K/E1371Q, although it too is somewhat weakened by the R334Q mutation (Fig. 10E). Login to comment 158 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys The strong effect of external Clon Pt(NO2)4 2-binding to R334Q/I344K/E1371Q channels is illustrated in Fig. 9D. Login to comment 159 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Because the effect of external SCN- on Pt(NO2)4 2- block was so dramatically altered in I344K/E1371Q (Fig. 7C, E, G), quantitative analysis of the effects of SCN- was also carried out (Fig. 11; Table 3). Login to comment 160 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys As for external Cl- (Fig. 8), there was a linear relationship between [SCN- ]o and Pt KD, both in E1371Q (Fig. 11A) and I344K/E1371Q (Fig. 11E), consistent with competition between SCN- and Pt(NO2)4 2- ions. Login to comment 161 ABCC7 p.Ile344Lys In I344K/ Fig. 5. Effect of mutations at the putative blocker binding site on [Cl- ]-dependence of block. Login to comment 162 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys (A) Relationship between Pt(NO2)4 2-binding affinity (Pt KD at 0 mV) and [Cl- ]o for E1371Q (black), K95Q/E1371Q (blue; see Fig. 1) and I344K/E1371Q (red; see Fig. 1). Login to comment 163 ABCC7 p.Glu1371Gln Asterisks indicate a significant difference from E1371Q (P b 0.001). Login to comment 165 ABCC7 p.Glu1371Gln Asterisks and daggers indicate a significant difference from E1371Q (asterisks, P b 0.005; daggers, P b 0.0000005). Login to comment 168 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys (A, B, E, F) Example macroscopic I-V relationships for R334Q/E1371Q (A, B) or R334Q/I344K/E1371Q (E, F) under high (154 mM; A, E) or low (4 mM; B, F) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of Pt(NO2)4 2- (at the concentrations indicated) to the intracellular solution. Login to comment 172 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln There was no significant difference in these parameters for R334Q/E1371Q (E) (P N 0.5). Login to comment 173 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Dotted lines indicate the fit to data for E1371Q (see Fig. 2H) or I344K/E1371Q (see Fig. 4J) as indicated. Login to comment 175 ABCC7 p.Glu1371Gln E1371Q, SCN K was greatly reduced (Fig. 11F; Table 3), Pt Kocc(0) was slightly increased (Fig. 11G; Table 3), and both SCN zb4; and Pt zb4;occ were increased (Fig. 11F, G; Table 3). Login to comment 176 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys In both E1371Q (Fig. 11D) and I344K/ E1371Q (Fig. 11H), SCN- binding had a greater destabilizing impact on Pt(NO2)4 2-binding than did Cl- . Login to comment 177 ABCC7 p.Ile344Lys 3.6. Effect on single channel conductance The results in Figs. 6 and 7 suggest that antagonistic ion:ion interactions inside the pore that are lost following removal of the positive charge at R334 are at least partially restored by the I344K mutation. Login to comment 178 ABCC7 p.Arg334Gln These interactions may be important for maximization of single channel conductance (see Introduction), as exemplified by the very low conductance observed for R334Q and for other substitutions at this site [33]. Login to comment 179 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys However, the reduced single channel conductance observed in R334Q channels (Fig. 12) was not significantly rescued by the I344K mutation (Fig. 12). Login to comment 180 ABCC7 p.Ile344Lys This contrasts with the previous observation that the I344K mutation, while having little influence on single channel conductance by itself, could almost completely restore the greatly diminished conductance seen when K95 in the inner vestibule is neutralized by mutagenesis [50]. Login to comment 182 ABCC7 p.Ile344Lys Fig. 7. Effect of other extracellular anions on Pt(NO2)4 2- block of I344K-containing channels. Login to comment 183 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys (A, B) Example macroscopic I-V relationships for I344K/E1371Q (A) or R334Q/I344K/E1371Q (B) with extracellular solution containing 150 mM SCN- . Login to comment 197 ABCC7 p.Glu1371Gln The present study using constitutively active E1371Q channels appears to effectively isolate the effects of Cl- acting inside the pore, at least for the test blocker Pt(NO2)4 2- . Login to comment 198 ABCC7 p.Glu1371Gln ABCC7 p.Arg899Gln Thus, block by cytoplasmic Pt(NO2)4 2- ions - and its dependence on extracellular [Cl- ] - is independent of the R899Q mutation (in an E1371Q background) (Fig. 2; Table 1). Login to comment 199 ABCC7 p.Arg899Gln This R899Q mutation has previously been shown to disrupt the antagonistic effects of Cl- and other extracellular anions on block by different cytoplasmic anions, including Pt(NO2)4 2- [45,46]. Login to comment 208 ABCC7 p.Glu1371Gln The ability of the E1371Q background effectively to isolate antagonistic interactions between external anions and intracellular Pt(NO2)4 2- blocking anions that occur inside the open channel pore affords a novel opportunity to investigate the nature and molecular bases of these antagonistic interactions. Login to comment 211 ABCC7 p.Ile344Lys Addition of a second positive charge in the inner vestibule in I344K strengthens Pt(NO2)4 2-binding (Figs. 4, 5, 8; Table 1), consistent with this residue also existing close to the internal blocker binding site [35,50] (Fig. 13A). Login to comment 216 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys (A-C) Effect of extracellular [Cl- ] ([Cl- ]o) on the measured Pt KD in E1371Q (A), K95Q/E1371Q (B) and I344K/E1371Q (C) at different membrane potentials as indicated in panel A. Straight-line fits to the data are to Eq. (3) as described in the Materials and methods. Login to comment 219 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment 221 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln Because of the lack of [Cl- ]o-dependence in R334Q/E1371Q, values for Pt Kocc, Pt zb4;occ, Cl K and Cl zb4; could not be obtained for this mutant. Login to comment 222 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Pt Kvac(0) (bc;M) Pt zb4;vac Pt Kocc(0) (bc;M) Pt zb4;occ Cl K (mM) Cl zb4; E1371Q 245 -0.39 1440 -0.63 179 +0.22 K95Q/E1371Q 1150 -0.23 4400 -0.58 296 +0.20 I344K/E1371Q 0.174 -0.42 978 -1.12 0.264 +1.09 R334Q/E1371Q 1120 -0.71 - - - - R334Q/I344K/E1371Q 31.8 -1.04 1500 -1.15 232 +0.23 Fig. 9. Effect of bound extracellular Cl-ions on the binding of intracellular Pt(NO2)4 2- ions. Login to comment 224 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment 233 ABCC7 p.Arg334Gln Indeed, the R334Q mutation significantly decreases Pt(NO2)4 2-binding affinity (Fig. 10C; Table 1). Login to comment 234 ABCC7 p.Lys95Gln Present results also suggest that the K95Q mutation causes a small decrease in the Cl-binding affinity of the outer site (Fig. 8F; Table 2). Login to comment 239 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys A second mechanism of knock-off in I344K-containing channel pores Addition of a second positive charge lining the inner vestibule in I344K/E1371Q increases intrinsic Pt(NO2)4 2-binding affinity ~1400-fold (Figs. 5, 8; Table 2), likely because the additional positive charge increases electrostatic interactions with divalent Pt(NO2)4 2- anions [47] (Fig. 13B). Login to comment 240 ABCC7 p.Ile344Lys However, this I344K mutation also has a strong influence Fig. 10. Login to comment 241 ABCC7 p.Arg334Gln Analysis of Pt(NO2)4 2- and Cl-binding to channel variants containing the R334Q mutation. Login to comment 242 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys (A, B) Effect of extracellular [Cl- ] ([Cl- ]o) on the measured Pt KD in R334Q/E1371Q (A) and R334Q/I344K/E1371Q (B) at different membrane potentials as indicated. Login to comment 246 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln Note that, because of the apparent lack of effect of external Clon Pt(NO2)4 2- block in R334Q/ E1371Q, it was not possible to obtain values for Pt Kocc or Cl K for this mutant. Login to comment 248 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment 251 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys (A, E) Effect of extracellular [SCN- ] ([SCN- ]o) on the measured Pt KD in E1371Q (A) and I344K/E1371Q (E) at 0 mV and -100 mV membrane potential. Login to comment 255 ABCC7 p.Glu1371Gln Dotted lines in F and G indicate the fit to data for E1371Q (from B and C). Login to comment 256 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment 259 ABCC7 p.Ile344Lys These two effects on binding of internal Pt(NO2)4 2- and external anions suggest that the I344K mutation alters, directly or indirectly, ion interactions with two different binding sites inside the pore. Login to comment 260 ABCC7 p.Ile344Lys The alternative to this - that I344K increases the anion binding affinity of a single site that interacts with both internal Pt(NO2)4 2- and external Cl- - can be ruled out based on the shape of the Pt KD-[Cl- ]o relationship for this mutant (Fig. 4J). Login to comment 263 ABCC7 p.Ile344Lys Consequently, if we assume that the I344K mutation is increasing the affinity of a Cl-binding site on the extracellular side of the Pt(NO2)4 2-binding site, then this could reflect an increase in the Cl- -binding affinity of the "normal" Cl-binding site in the outer vestibule, or the introduction of a novel Cl-binding site elsewhere in the pore. Login to comment 266 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys However, the R334Q mutation has only a minor impact on Cl-binding in the presence of I344K, and Cl-binding to the R334Q/I344K/E1371Q mutant remains strong and strongly voltage-dependent (Fig. 10; Table 2). Login to comment 267 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys This suggests that in I344K-containing channels (unlike E1371Q background channels), significant binding of extracellular Cl- occurs at a site that is independent of R334. Login to comment 268 ABCC7 p.Arg334Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Indeed, knock-off of Pt(NO2)4 2- by Cl- appears similar in I344K/ E1371Q and R334Q/I344K/E1371Q channels (Fig. 10D), suggesting that anion binding near R334 plays little role in ion:ion interactions that occur in channels bearing the I344K mutation. Login to comment 269 ABCC7 p.Ile344Lys Furthermore, the voltage-dependence of external Cl-binding is greatly increased in I344K-containing channels. Login to comment 271 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys The apparent anion selectivity of the external anion binding site is also slightly altered in I344K-containing channels (Fig. 7G, H), and this appears independent of the presence or absence of the R334Q mutation. Login to comment 272 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Not only the binding affinity of external Cl- and internal Pt(NO2)4 2- , but also the Table 3 Mean parameters obtained from analysis of the [SCN- ]o-dependence of Pt(NO2)4 2- block in E1371Q and I344K/E1371Q, as described in Fig. 11. Login to comment 274 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys Pt Kocc(0) (bc;M) Pt zb4;occ SCN K (bc;M) SCN zb4; E1371Q 17,500 -0.54 14,000 +0.137 I344K/E1371Q 32,900 -0.94 1.95 +0.917 Fig. 12. Login to comment 275 ABCC7 p.Arg334Gln Single channel conductance of R334Q-containing channels. Login to comment 276 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys (A) Example single-channel currents recorded at a membrane potential of -60 mV for the wild type, R334Q, and R334Q/I344K channels as indicated. Login to comment 282 ABCC7 p.Arg334Gln ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys strengthoftheinteractionbetweenthem,isalteredinI344K-containingchan- nels.Thus,thedestabilizingeffectofCl- onPt(NO2)4 2-binding-evaluated fromthedifferencebetweenPt(NO2)4 2- bindinginvacantandCl- -occupied channels(i.e.betweenPt KvacandPt Kocc)-ismuchgreaterinI344K/E1371Q (and to a lesser extent R334Q/I344K/E1371Q) than in E1371Q or K95Q/ E1371Q(Fig.9).Strengthenedinteractionsbetweenboundanionsarealso suggestedbytheincreasedapparentcouplingbetweenthemovementof Pt(NO2)4 2- andCl- ionsinsidetheporeinI344K(Fig.8E). Login to comment 283 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys One model that appears consistent with these results is that addition of a second positive charge in the inner vestibule (in I344K/E1371Q) increases the number of anions that can bind within this region of the pore (Fig. 13B). Login to comment 287 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys These different mechanisms are illustrated in wild type (A, C) and R334Q/I344K channels (B, D) that are proposed to isolate the two different mechanisms of ion:ion interaction; it is presumed that both mechanisms are active in I344K (and neither in R334Q). Login to comment 289 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys (B) In R334Q/I344K channels, Cl- does not bind to the outer pore mouth, but instead binds further into the pore from its extracellular end (experiencing a greater fraction of the transmembrane electric field) and binds close to the introduced lysine in the inner vestibule, where it strongly antagonizes Pt(NO2)4 2-binding (leading to very strong [Cl- ]o-dependence of block). Login to comment 291 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys (D) In R334Q/I344K, even if multiple Cl-ions are able to bind close together in the inner vestibule, this does not result in rapid Cl-permeation in the absence of Cl-binding to the outer pore region. Login to comment 293 ABCC7 p.Ile344Lys nearby sites could then introduce a novel, strongly destabilizing effect of external Clon internal Pt(NO2)4 2-binding in I344K-containing channels. Login to comment 295 ABCC7 p.Glu1371Gln Furthermore, because this destabilizing effect involves local interactions between bound anions (Fig. 13B), it may be relatively independent of anion binding in the outer vestibule of the pore - close to R334, which is required for normal anion:anion interactions observed in E1371Q background channels (Fig. 13A). Login to comment 297 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys In effect, in the R334Q/I344K double pore mutant, one kind of antagonistic Cl- :blocker interaction (Fig. 13A) has been replaced by another with a different molecular mechanism (Fig. 13B). Login to comment 298 ABCC7 p.Ile344Lys Presumably in I344K channels both knock-off mechanisms shown in Fig. 13A, B are in effect. Login to comment 300 ABCC7 p.Glu1371Gln ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Understanding the observed properties of Pt(NO2)4 2- block of I344K channels Block of I344K/E1371Q by cytoplasmic Pt(NO2)4 2-was previously described as being of very high affinity, very strong voltage dependence, and very high sensitivity to extracellular [Cl- ] [47]. Login to comment 311 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Ile344Lys Reduced conductance in K95Q is restored in K95Q/I344K, leading to the proposal that a positive charge located at either of these two nearby residues is able to support physiologically important Cl-binding in the inner vestibule [35, 50]. Login to comment 312 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys However, although I344K appears capable of restoring sensitivity to external Cl-ions that is lost in R334Q, this does not result in a significant restoration of unitary Cl-conductance (Fig. 12). Login to comment 313 ABCC7 p.Arg334Gln ABCC7 p.Ile344Lys In other words, although it is suggested that in R334Q/I344K channels one form of knock-off inside the pore has been replaced by another (Fig. 13B), this surrogate knock-off mechanism does not appear to be capable of supporting high Cl-conductance in the way that normal ion-ion interactions in wild type CFTR are proposed to do (Fig. 13C, D). Login to comment