ABCMdb

PMID: 24671572

Linsdell P
State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore.
Pflugers Arch. 2014 Dec;466(12):2243-55. doi: 10.1007/s00424-014-1501-7. Epub 2014 Mar 28., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Lys95Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4 2- altered blocker effects both on Cl-permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4 2- interaction with a single site within the pore. Login to comment 31 ABCC7 p.Ser1141Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Furthermore, when the number of positive charges lining this part of the inner vestibule is increased from one (as in wild type) to two (for example in the channel mutants I344K, V345K, and S1141K), blocker potency is increased [8, 36]. Login to comment 42 ABCC7 p.Glu1371Gln Some experiments were carried out with channels containing the NBD2 mutation E1371Q, which this laboratory [25-27, 29, 36] and others [2, 3] have used to abolish ATP-dependent channel gating and lock CFTR channels in the open state. Login to comment 43 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Where the properties of different channel pore variants (wild type, K95Q, I344K, V345K) have been directly compared in wild type and E1371Q backgrounds, the wild-type background is referred to as "1371E" to indicate that the endogenous glutamate residue is present at this position. Login to comment 53 ABCC7 p.Lys95Gln Under most circumstances nH was very close to unity, except for experiments using K95Q mutant channels (Figs. 3(B) and 4(B)) where it was in the range 0.43-0.63 (see, for example, Fig. 3(B)). Login to comment 61 ABCC7 p.Glu1371Gln (F) Example leak-subtracted macroscopic I-V relationships for E1371Q-CFTR in the presence of 1 mM ATP, 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 62 ABCC7 p.Glu1371Gln (G, H) Mean data from experiments with E1371Q channels under the same conditions used for wild-type channels in (C-E). Login to comment 63 ABCC7 p.Glu1371Gln (I-K) Mean values of KD (at 0 mV in I and at +50 mV in J) and zb4; (K) obtained from fits of KD-Vrelationships such as those shown in (E) and (H), for both wild type and E1371Q channels under different nucleotide conditions as indicated. Login to comment 76 ABCC7 p.Glu1371Gln (C) Mean effect of this same concentration of Pt(NO2)4 2- on macroscopic current amplitudes under different conditions (black filled circle, wild type with 1 mM ATP; white empty circle, wild type with 1 mM ATP plus 2 mM PPi; filled down-pointing triangle E1371Q with 1 mM ATP), estimated from experiments such as those shown in Fig. 1. Login to comment 92 ABCC7 p.Glu1371Gln Block was also strongly voltage dependent when the channel open state was stabilized using the E1371Q mutation (Fig. 1(F-H)), which results in channel open probabilities close to one under these conditions [27]. Login to comment 93 ABCC7 p.Glu1371Gln Furthermore, in the E1371Q mutant, the blocking effects of Pt(NO2)4 2- were independent of either ATP concentration or the addition of 2 mM PPi (Fig. 1). Login to comment 95 ABCC7 p.Glu1371Gln Block of E1371Q was strongly voltage dependent under all conditions (Fig. 1(K)). Login to comment 98 ABCC7 p.Glu1371Gln The strongly voltage-dependent effects of this concentration of Pt(NO2)4 2- on unitary current amplitude (Fig. 2(B)) closely resemble the effects of this concentration of blocker on macroscopic wild-type currents following treatment with PPi, or in E1371Q mutant channels (Fig. 2(C, D)), whereas they are considerably weaker and more voltage-dependent than effects on macroscopic wild-type currents in the absence of PPi (Fig. 2(C, D)). Login to comment 106 ABCC7 p.Glu1371Gln When channel closure is inhibited using PPi or the E1371Q mutation, the effect on Cl-permeation is effectively isolated (Figs. 1 and 2). Login to comment 108 ABCC7 p.Glu1371Gln The effects of mutations in this region on the open-channel blocking effect of Pt(NO2)4 2- , isolated using E1371Q-containing channels, are shown in Fig. 3. Login to comment 109 ABCC7 p.Lys95Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Removal of the key endogenous positive charge (using the K95Q mutation) greatly weakens Pt(NO2)4 2- block (Fig. 3(B-D)), whereas addition of a second pore-lining positive charge (in I344K or V345K) dramatically strengthens open-channel block (Fig. 3(B-D)). Login to comment 110 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Interestingly, not only blocker binding affinity (Fig. 3(D)) but also blocker voltage dependence (Fig. 3(E)) appeared correlated with the number of positively charged lysine side chains lining the inner vestibule of the pore; both I344K/E1371Q and V345K/E1371Q gave very strong, very strongly voltage-dependent block (Fig. 3(A, C-E)). Login to comment 111 ABCC7 p.Glu1371Gln To further investigate the relationship between Pt(NO2)4 2- block and gating, block of macroscopic currents was compared in 1371E (wild type) and E1371Q backgrounds (Fig. 4). Login to comment 112 ABCC7 p.Lys95Gln ABCC7 p.Glu1371Gln The weak blocking effects of Pt(NO2)4 2- on K95Q-containing channels appeared independent of the presence of the E1371Q mutation (Fig. 4(B)), suggesting that no high-affinity Pt(NO2)4 2-binding site exists outside of the channel pore. Login to comment 113 ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys In contrast, the strong inhibitory effects of Pt(NO2)4 2- on both I344K- and V345K-containing channels were observed in both E1371Q and 1371E backgrounds (Fig. 4(B)). Login to comment 114 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys ABCC7 p.Ile344Lys Most strikingly, however, the very strong voltage dependence of block seen in both I344K/E1371Q and V345K/E1371Q (Fig. 3) was not observed in I344K/1371E and V345K/1371E channels that were allowed to open and close normally; instead, block was very strong but almost completely voltage-independent (Fig. 4). Login to comment 115 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys In many ways, these results with I344K and V345K recapitulate the results observed when comparing wild type and E1371Q channels (Fig. 1); block is weaker in E1371Q-containing channels (Fig. 4(C)), and blocker voltage dependence is greatly decreased (Fig. 4(D)). Login to comment 116 ABCC7 p.Glu1371Gln In fact, the discrepancy between 1371E and E1371Q channels, both in blocker affinity (Fig. 4(E)) and blocker voltage dependence (Fig. 4(F)), was greater when more fixed positive charges were present in the pore inner vestibule. Login to comment 118 ABCC7 p.Lys95Gln Because of its very low single-channel conductance [8], gating of K95Q channels was not investigated; results shown in Fig. 4(B) suggest that Pt(NO2)4 2- should not affect the gating of these channels. Login to comment 119 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Gating of I344K and V345K channels was affected by Pt(NO2)4 2- in a similar manner to those of wild-type channels, but at much lower concentrations. Login to comment 122 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Investigation of the effect of different concentrations of Pt(NO2)4 2- on the PO of wild type, I344K and V345K channels (Fig. 5(C)) confirms a dramatic increase in the apparent potency with which Pt(NO2)4 2- ions interact with these channel pore mutants. Login to comment 123 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Effect of extracellular Clon Pt(NO2)4 2- block As a consequence of the striking difference in voltage-dependence of block of I344K and V345K-containing channels studied in 1371E (Fig. 4) or E1371Q backgrounds (Fig. 3), there was an unusual "cross-over" effect in the relationship between the KD for Pt(NO2)4 2- block and voltage (Fig. 4(B)), suggesting that block is stronger in 1371E than in E1371Q channels at positive voltages, but weaker in 1371E at the most negative voltages studied. Login to comment 126 ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Under these conditions, cross-over of the KD-voltage relationship was observed for wild type and I344K channels, whereas for V345K channels block was now stronger in E1371Q channels at all voltages studied. Login to comment 128 ABCC7 p.Glu1371Gln (A) Example leak-subtracted macroscopic I-V relationships for the different E1371Q-containing CFTR constructs named, recorded before (control) and after the addition of Pt(NO2)4 2-to the intracellular solution at the concentrations indicated. Login to comment 131 ABCC7 p.Glu1371Gln (D, E) Mean values of KD (at 0 mV) and zb4; obtained from fits of KD-V relationships such as those shown in (C), as a function of the number of positively charged lysine side chains lining the inner vestibule of the pore in different channel constructs. Asterisks indicate a significant difference from wild type (E1371Q) (P<0.005). Login to comment 133 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln However, whereas the Pt(NO2)4 2- -induced decrease in PO could easily be studied at the single-channel level at depolarized voltages at which open-channel blocking effects are practically absent (Figs. 2 and 5), the discrepancy between the KD measured in 1371E and E1371Q channels shown in Fig. 6(C) was most striking at hyperpolarized voltages at which effects on single-channel current amplitudes (as reflected by effects on E1371Q channels) are expected to be very strong, and could not therefore be investigated directly using single-channel recordings. Login to comment 135 ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Under these ionic conditions, the E1371Q mutation increased the apparent affinity of block (Fig. 7(A, B)), an effect that was especially strong in I344K and V345K channels and at hyperpolarized voltages. Login to comment 136 ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys This contrasts with what was found under high [Cl- ] conditions, where the E1371Q mutation decreased the affinity of block, especially in I344K and V345K (Fig. 4(C)). Login to comment 137 ABCC7 p.Glu1371Gln The E1371Q mutation also increased the voltage dependence of block under low [Cl- ] Fig. 4 Gating-dependent Pt(NO2)4 2- block in pore mutant forms of CFTR. Login to comment 139 ABCC7 p.Glu1371Gln (B) Mean KD values obtained from such data as described in Figs. 1 and 3 (white empty circle), compared to data from the same channel mutants in an E1371Q background (black filled circle) (taken from Fig. 3(C)). Login to comment 140 ABCC7 p.Glu1371Gln (C, D) Comparison of mean KD (at 0 mV) and zb4; values estimated for different channel variants in an E1371Q background (black bars) versus a 1371E background (grey bars). Login to comment 141 ABCC7 p.Glu1371Gln Asterisks indicate a significant difference from the same channel variant in an E1371Q background (P<0.0005). Login to comment 142 ABCC7 p.Glu1371Gln (E, F) Direct quantification of the effect of different E1371 background on KD (at 0 mV) and zb4;, evaluated as the mean value in a 1371E background as a fraction of that in an E1371Q background, and plotted relative to the number of positively charged lysine side chains lining the inner vestibule of the pore in different channel constructs. Asterisks indicate a significant difference from the value estimated for wild type (P<0.05). Login to comment 146 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys However, [Cl- ] has little effect on the voltage dependence of block (Fig. 7(E)), which is already very strong in I344K/E1371Q and V345K/E1371Q channels (Figs. 4(D) and 7(C)). Login to comment 148 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys The strong effect of [Cl- ] on the strength of block of I344K and V345K is abolished (Fig. 7(D)), and high [Cl- ] actually reduces the strong apparent voltage dependence of block in these same mutants (Fig. 7(E)). Login to comment 150 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys (A) Example currents recorded from inside out patches each containing two active CFTR channels, either I344K (top) or V345K (bottom). Login to comment 152 ABCC7 p.Val345Lys All closed state is indicated by the line to the left. Note that V345K exhibits a significantly reduced single-channel current amplitude, as described and quantified previously [8]. Login to comment 155 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys (C) Effect of different concentrations of Pt(NO2)4 2- on the PO of wild type, I344K and V345K channels as indicated. Login to comment 159 ABCC7 p.Glu1371Gln Multiple effects of Pt(NO2)4 2- on conductance and gating The open-channel blocking effects of Pt(NO2)4 2are the easiest to isolate and observe-these effects can be seen from the effect of Pt(NO2)4 2- on single-channel current amplitude (Fig. 2(A, B)) and are also isolated in channels in which the open state has been stabilized, either using PPi to interrupt normal ATP-dependent gating (Fig. 1) or using the ATP hydrolysis-defective E1371Q mutant (Figs. 1, 3, and 6). Login to comment 162 ABCC7 p.Glu1371Gln (A, B) Example leak-subtracted macroscopic I-V relationships for the different CFTR constructs named, either in an E1371Q background (A) or a 1371E background (B), with a low (4 mM) [Cl- ] extracellular solution. Login to comment 165 ABCC7 p.Lys95Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Mean of data from 5 to 11 patches Pt(NO2)4 2-binding within the inner vestibule of the pore, since they are weakened by the K95Q mutation and strengthened in I344K and V345K (Fig. 3(C)), as described previously [8, 36]. Login to comment 172 ABCC7 p.Lys95Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Thus, effects on gating are apparently lost in K95Q (Fig. 4(B)), and are substantially strengthened by mutations (I344K, V345K) that increase the strength of binding inside the pore (Figs. 4 and 5). Login to comment 175 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys However, these two effects appear unable to explain the overall effects of Pt(NO2)4 2- in I344K and V345K (Figs. 4(B) and 6(C)), or effects on wild type under low extracellular [Cl- ] conditions (Fig. 6(C)). Login to comment 176 ABCC7 p.Glu1371Gln In each of these situations, block at very negative voltages appears anomalously weak in 1371E channels when compared to E1371Q, hinting that Pt(NO2)4 2- block might actually be associated with an increase in PO under these conditions. Login to comment 181 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln In each panel, black bars represent an E1371Q background and grey bars a 1371E background; asterisks indicate a significant difference from the same channel variant in an E1371Q background (P<0.05); and (in D and E) daggers represent a significant difference between high and low external [Cl- ] conditions (P<0.05). Login to comment 187 ABCC7 p.Lys95Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Perhaps supporting the former hypothesis, this putative stimulatory effect of Pt(NO2)4 2- does appear to be positively correlated with strong open-channel block, i.e., it is not observed in K95Q but is prominent in I344K and V345K channels that show strong binding, and at negative voltages that promote open-channel block (Fig. 6). Login to comment 193 ABCC7 p.Glu1371Gln Effects on open channels are isolated in E1371Q channels. Login to comment 195 ABCC7 p.Glu1371Gln Inhibitory effects on PO are due to effects on closed channels (increasing interburst duration), and so are most prominent in macroscopic current recordings under conditions in which open-channel block is relatively weak (e.g., at positive voltages), leading to the largest discrepancy in overall blocking effects between 1371E and E1371Q channels (Figs. 1, 2(C), and 4(B)). Login to comment 198 ABCC7 p.Glu1371Gln Effect of extracellular Cl- Increasing extracellular [Cl- ] weakens Pt(NO2)4 2- block of open E1371Q channels (Fig. 7(D)), as described previously for wild-type CFTR [11, 35]. Login to comment 200 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Interestingly, this apparent knock-off effect is greatly strengthened in I344K/E1371Q and V345K/E1371Q (Fig. 7(D)), suggesting Fig. 8 Schematic summary of the effects of Pt(NO2)4 2- . Login to comment 203 ABCC7 p.Glu1371Gln Experiments carried out on E1371Q channels are assumed to isolate effects on open channels (to the right of the dotted line in (A)). Login to comment 206 ABCC7 p.Glu1371Gln Block of macroscopic 1371E channels is generally stronger than that of E1371Q channels at high extracellular [Cl- ] (Fig. 1(E) and 4(B)), whereas block is generally weaker in 1371E channels at low extracellular [Cl- ] (Fig. 6(C)). Login to comment