ABCMdb

ABCC7 p.Ser1141Lys

None has been submitted yet.

Publications

PMID: 20142516 [PubMed] Zhou JJ et al: "Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore."

No. Sentence Comment

16 Thus, the mutant channel K95S/S1141K showed Cl conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Login to comment
19 This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). Login to comment
20 The S1141K mutant had similar Cl conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO2)4 2 in inside-out membrane patches. Login to comment
21 Furthermore, in cell-attached patch recordings, apparent voltage- -dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Login to comment
62 Fig. S1 shows the inhibitory effects of TLCS and lonidamine on wild type, K95S, K95S/S341K, and K95S/S1141K-CFTR. Login to comment
64 Fig. S3 shows the apparent time-and voltage-dependent inhibition of S1141K by intracellular ATP in inside-out patches. Login to comment
66 Fig. S5 shows the block of wild type, S1141K, and S341K by intracellular NPPB. Login to comment
67 Fig. S6 shows the block of E1371Q and E1371Q/S1141K by intracellular Pt(NO2)4 2 ions. Login to comment
76 In a K95S background, the introduction of a positive charge in either TM6 (S341K) or TM12 (S1141K) led to a significant increase in the apparent potency of NPPB block compared with K95S alone (Fig. 1), with mean Kd(0) values of 35.8 ± 2.0 µM (n = 4) in K95S/S341K and 10.5 ± 1.8 µM (n = 4) in K95S/S1141K (Fig. 1 D), again with no significant change in apparent voltage dependence of block (z of 0.17 ± 0.02 [n = 4] in K95S/ S341K and 0.22 ± 0.03 [n = 4] in K95S/S1141K). Login to comment
77 In fact, in the K95S/S1141K mutant, the apparent Kd was not significantly different than that observed in wild type (P > 0.4; Fig. 1 D), suggesting that the role played by the positive charge at position 95 in the interaction between NPPB and the pore can be completely recovered by moving this positive charge from TM1 to TM12. Login to comment
98 As with the open-channel blocker experiments described above, the introduction of a positive charge in TM12 led to a significant recovery of wild-type pore properties-in this case, a dramatic increase in unitary conductance in the K95S/S1141K double mutant compared with K95S alone (Fig. 2). Login to comment
99 In the K95S background, the second site mutation (S1141K) The positive charge at K95 is also important for attracting Cl ions into the pore, and removal of this charge by mutagenesis is associated with a dramatic decrease in unitary Cl conductance (Ge et al., 2004). Login to comment
106 , wild type (B); , K95S (B and C); , K95S/S341K (C); , K95S/S1141K (C). Login to comment
107 Each set of data has been fit by Eq. 1, giving for wild-type: Kd(0) = 12.3 ± 0.1 µM and z = 0.20 ± 0.01; for K95S: Kd(0) = 83.9 ± 0.6 µM and z = 0.16 ± 0.00; for K95S/S341K: Kd(0) = 33.7 ± 0.7 µM and z = 0.15 ± 0.01; and for K95S/S1141K: Kd(0) = 10.3 ± 0.1 µM and z = 0.22 ± 0.01. Login to comment
111 Interestingly, the S1141K mutation alone led to a small but significant reduction in unitary conductance compared with wild type, to 8.02 ± 0.08 pS restored conductance at hyperpolarized voltages to 6.28 ± 0.06 pS (n = 9), 75% of wild-type values, and at depolarized voltages to 5.37 ± 0.04 pS (n = 10), 67% of Figure 2. Single-channel conductance is restored by moving a positive charge from TM1 to TM12. Login to comment
116 Each has been fitted by the sum of two Gaussian functions with mean amplitudes of 0 pA and at +50 mV: 0.397 pA (wild type), 0.046 pA (K95S), 0.266 pA (K95S/S1141K), and 0.331 pA (S1141K); at 50 mV: 0.401 pA (wild type), 0.058 pA (K95S), 0.349 pA (K95S/S1141K), and 0.437 pA (S1141K). Login to comment
118 (C and D) Mean single-channel I-V relationships for wild-type (C, ), K95S (C and D, ), K95S/S1141K (D, ), and S1141K (D, ). Login to comment
121 Daggers indicate a significant difference from wild type (P < 1010 for both K95S and K95S/S1141K; P < 0.05 for S1141K). Login to comment
167 K95C/S1141C channel investigated the S1141K mutant at the macroscopic current level using depolarizing voltage ramp protocols like those used in Fig. 1, it became apparent that channel function had been altered in a way we had not anticipated from our initial single-channel experiments (see Fig. 2). Login to comment
168 With a low extracellular Cl concentration, macroscopic currents in S1141K showed outward rectification leading to a flattening of the I-V relationship at hyperpolarized voltages (Fig. 5, A and B). Login to comment
170 We were therefore interested to know how the presence of two adjacent positive charges in this region of the inner vestibule-as presumably exist in the S1141K single mutant-would influence interactions with open-channel blockers. Login to comment
178 Replacing the anionic TES buffer with the cationic pH buffer Tris did not alter the unusual shape of the I-V relationship in S1141K (not depicted). Login to comment
179 In contrast, altering the ATP concentration did lead to striking time-and voltage-dependent changes in current amplitude that suggest an inhibitory effect of ATP on S1141K (Fig. S3). Login to comment
183 As a result, although PPi stimulated macroscopic current amplitude in S1141K at depolarized voltages, it actually inhibited current at the most hyperpolarized voltages studied (Fig. 5 D). Login to comment
184 None of these effects was observed in wild type or K95S/S1141K, two channel variants with a single positive charge in this part of the inner vestibule of the pore (Fig. 5), or in S341K (not depicted). Login to comment
185 Because the S1141K mutant did not strongly affect either unitary conductance or the linearity of the I-V relationship under symmetrical high Cl conditions (Fig. 2), we considered that the unusual I-V shape shown in Fig. 5 might reflect voltage-dependent block of S1141K by some negatively charged substance present in our intracellular solutions. Login to comment
186 However, the solutions Figure 5. Apparent inhibition of S1141K at hyperpolarized voltages. Login to comment
189 (B and C) Relative shape of the I-V relationship in the presence of 1 mM ATP (B) or 1 mM ATP plus 2 mM PPi (C), analyzed by plotting the current at each voltage relative to the current amplitude at 0 mV, for wild type (), S1141K (), and K95S/S1141K (). Login to comment
191 Note that PPi causes a voltage-independent stimulation in wild type () and K95S/S1141K (), whereas in S1141K (), PPi causes stimulation at depolarized voltages and inhibition at hyperpolarized voltages. Login to comment
192 Asterisk indicates the voltage range over which the effects of PPi on wild type and S1141K were significantly different (P < 0.05). Login to comment
193 Mean of data from four to five patches in B-D. tion of 10 mM ATP caused a potent inhibition of S1141K/E1371Q current during 400-ms hyperpolarizing voltage steps from a holding potential of +60 mV (Fig. 6 A). Login to comment
197 In contrast to the strong inhibition of S1141K/ E1371Q current by ATP under these conditions, 10 mM prolongation of CFTR channel open times (Vergani et al., 2003; Gadsby et al., 2006; Stratford et al., 2007). Login to comment
199 Although the reasons for this constitutive activity, which contrasts with a complete lack of spontaneous activity we observe for other CFTR constructs expressed in BHK cells, are unknown, it did allow us to quantify ATP effects on S1141K current amplitude in an E1371Q background in inside-out membrane patches, beginning with 0 ATP control conditions (Fig. 6). Login to comment
200 With a low extracellular Cl concentration (4 mM), the addi- Figure 6. Inhibition of S1141K/E1371Q-CFTR by intracellular ATP. Login to comment
201 (A) Example macroscopic currents carried by S1141K/E1371Q during hyperpolarizing voltage steps to between +60 and 100 mV recorded under conditions of low extracellular Cl concentration (4 mM). Login to comment
205 Also shown are the effects of 10 mM ATP on E1371Q () and K95S/S1141K/E1371Q () at 100 mV. Login to comment
206 (C) Example macroscopic S1141K/E1371Q currents during voltage steps to between +60 and 100 mV recorded with a high extracellular Cl concentration (154 mM) before (control) and after the addition of 10 mM Na2ATP to the intracellular solution in the absence of PKA. Login to comment
218 Outward rectification in cell-attached patches under these conditions was even more pronounced in S1141K/ E1371Q, and again this rectification was relieved by excision of the membrane patch, resulting in a linear I-V relationship in inside-out patches. Login to comment
221 Such analysis revealed that current inhibition in cell-attached patches was significantly stronger in S1141K/E1371Q than in E1371Q at hyperpolarized voltages, both at high extracellular Cl concentrations (Fig. 7 C) and at low Cl concentrations (Fig. 7 D). Login to comment
224 However, the effects of complementary mutations at K95 and at S1141 in TM12 suggest that the important functional role of this positive ATP had no effect on E1371Q and only a very small inhibitory effect on K95S/S1141K/E1371Q (Fig. 6 B). Login to comment
225 Interestingly, the inhibition of S1141K/E1371Q by intracellular ATP was very much weaker when using a high extracellular Cl concentration (154 mM; Fig. 6 C) during voltage steps of the same duration. Login to comment
232 The apparent voltage-dependent block of S1141K by ATP, and likely also by PPi (Fig. 5), complicated studies of the interaction of this mutant with open-channel blockers like those studied in Fig. 1 and Fig. S1. Login to comment
233 Nevertheless, we were able to study NPPB block of S1141K under conditions where ATP block was weak (by using symmetrical Cl conditions) and in the absence of PPi. Login to comment
234 Under these conditions, a low concentration of NPPB (10 µM) had significantly more potent blocking effects on S1141K than on wild type (Fig. S5). Login to comment
235 The novel apparent inhibitory effects of ATP and PPi seen in S1141K but not in wild type suggest that this mutant might be particularly susceptible to block by polyvalent anions. Login to comment
237 Under ATP-free conditions, block by intracellular Pt(NO2)4 2 was dramatically more potent in E1371Q/S1141K compared with E1371Q alone, leading to a 38-fold decrease in mean Kd(0) (Fig. S6). Login to comment
238 This result is consistent with the S1141K mutation preferentially increasing the strength of interactions between polyvalent anions and the pore. Login to comment
239 Adding an extra positive charge to the inner vestibule of the pore decreases channel current in intact cells Because S1141K shows increased sensitivity to inhibition by intracellular anions such as ATP (Fig. 6), PPi (Fig. 5), NPPB (Fig. S5), and Pt(NO2)4 2 (Fig. S6), we wondered if this sensitivity would result in inhibition of channel currents in intact cells. CFTR channel currents are known to be subject to voltage-dependent inhibition by unknown anions present in the cytosol (Tabcharani Figure 7. Enhanced voltage-dependent inhibition in cell-attached patches in S1141K-CFTR. Login to comment
240 (A) Example macroscopic currents carried by E1371Q and S1141K/E1371Q-CFTR in cell-attached patches (left panels) after excision into the inside-out patch configuration (middle panels) and after the addition of 10 µM CFTRinh-172 to the intracellular solution (right panels). Login to comment
248 The ability of a positive charge located in TM1 (K95), TM6 (S341K), or TM12 (S1141K) to support blocker interactions is consistent with each of these TMs influencing the movement of blocking anions in the pore. Login to comment
259 The similarity of wild type and K95S/ S1141K in terms of single-channel conductance (Fig. 2) and interactions with open-channel blockers (Fig. 1 and Fig. S1) suggests that these two residues are almost completely interchangeable in functional terms. Login to comment
263 Thus, we suggest that TM1 and TM12 are located close together in the inner vestibule of the pore, such that the K95S/S1141K double mutant involves transplantation of fixed positive charge over a short distance. Login to comment
271 This would not be surprising in evolutionary terms because maximization of Cl conductance is the physiologically meaningful role of the positive charge associated with K95 in this part of the inner vestibule of the pore, with interaction with channel blockers current amplitude in cell-attached patches as a fraction of current in the same patch after excision into the inside-out patch configuration for both E1371Q () and S1141K/E1371Q (), with 154 mM Cl (C) or 4 mM Cl (D) in the extracellular solution. Login to comment
275 This first became apparent to us as a novel sensitivity to block by intracellular ATP molecules (Fig. 6), although it appears that S1141K also increases sensitivity to PPi (Fig. 5), NPPB (Fig. S5), and Pt(NO2)4 2 (Fig. S6). Login to comment
276 Block by intracellular ATP appears as a voltage- and extracellular [Cl ]-dependent phenomenon (Fig. 6), suggesting that negatively charged ATP molecules are attracted into the pore of S1141K-CFTR by the increased number of fixed positive charges in the inner vestibule of this mutant. Login to comment
282 Given our uncertainty concerning which species of ATP are responsible for block of S1141K, it is difficult to draw conclusions on the relative effect of an additional fixed positive charge at this position on interactions with monovalent versus polyvalent anions. Login to comment
284 Consistent with this, the apparent affinity of block by small, divalent Pt(NO2)4 2 anions was increased 38-fold in S1141K/ E1371Q compared with E1371Q (Fig. S6). Login to comment
285 In contrast, S1141K had a more modest effect on block by the larger, monovalent organic anion NPPB (Fig. S5), with the apparent Kd for this blocker being increased less than fourfold under different experimental conditions. Login to comment
286 Based on this highly limited survey of blocking anions, it seems reasonable to suggest that the additional positive charge present in the pore of S1141K channels favors interactions with polyvalent anions. An analogous situation has been described in K+ channel pores, where fixed negative charges in the channel protein preferentially attract polyvalent cationic blockers into the pore compared with monovalent K+ ions, leading to channel block and current rectification (Zhang et al., 2006). Login to comment
291 Interestingly, in all cases, blocker apparent valence was not significantly different between wild type and double mutants showing restored blocker binding (K95S/S341K, K95S/S1141K), suggesting that blocker movement in the TM electric field was well conserved in these mutants. Login to comment
294 The apparent functional interchangeability and physical proximity of K95 and S1141 allowed us to explore the consequences of increasing the number of positive charges in this region of the inner vestibule of the pore apparently available to interact with cytoplasmic anions from one (as we assume to exist in wild type) to two (in the S1141K mutant). Login to comment
295 Although increasing the number of positive charges in a localized region of the inner vestibule of the pore (according to this model) from 0 (in K95S) to one (wild type and K95S/S1141K) was associated with a dramatic increase in Cl conductance (Fig. 2), increasing further to two positive charges (S1141K) actually led to a slight decrease in conductance (Fig. 2). Login to comment
298 Although S1141K was no better than wild type in terms of Cl conductance, it did show an increased We thank Dr. David Gadsby for providing the cys-less CFTR cDNA. Login to comment
340 Although block appears strong in wild type (E1371Q), it is still significantly strengthened in S1141K (Fig. 7), suggesting that the number of fixed positive charges in the inner vestibule of the pore controls interactions with endogenous cytoplasmic-blocking molecules, and therefore (in intact cells) overall channel function in terms of the rate of anion efflux. Login to comment
343 This may be because the presence of an additional positive charge favors attraction of polyvalent anions into the pore, whereas the normal substrates of CFTR-mediated transport (Cl and HCO3  ) are monovalent anions. An overall decrease in channel function in intact cells is demonstrated in on-cell current recordings (Fig. 7), which show decreased Cl currents (relative to unblocked currents after patch excision to the inside-out configuration) at hyperpolarized voltages in S1141K/E1371Q relative to E1371Q alone. Login to comment

PMID: 23083715 [PubMed] El Hiani Y et al: "Tuning of CFTR chloride channel function by location of positive charges within the pore."

No. Sentence Comment

14 Thus, although neutralization of K95 causes a decrease in both Cl conductance and sensitivity to cytoplasmic open-channel blockers, these changes in channel function can be reversed by concurrent mutagenesis of a serine residue in TM12 to lysine (the S1141K mutant), suggesting that these two nearby residues lining the inner vestibule are functionally interchangeable (8). Login to comment
15 Interestingly, using the S1141K mutation to introduce a second positive charge to the inner vestibule did not increase Cl conductance (suggesting that a single positive charge is sufficient to maximize conductance), but instead conferred strong block by cytoplasmically applied multivalent anions (leading to an overall decrease in channel function) (8). Login to comment
57 Similar results were previously reported for NPPB block of K95S/S341K and K95S/S1141K (8). Login to comment
58 A striking and interesting effect of adding a second positive charge to the inner vestibule, previously demonstrated in S1141K (8), was a dramatic increase in susceptibility to block by polyvalent anions present in the cytoplasmic solution. Login to comment
90 However, although a single positive charge is necessary, the addition of a second positive charge to this region of the pore (as in the point mutants Q98K, I344K, V345K, M348K, and A349K) failed to increase conductance above WT levels (Fig. 2), as previously observed for S1141K (8). Login to comment
106 It was previously reported that the double mutant K95S/S1141K showed slightly increased potency of NPPB block compared with WT (8). Login to comment
146 Again this appears to be a relatively nonsite-specific effect of positive charge, since all mutants studied (Q98K, S341K, I344K, V345K, M348K, and A349K) led to significant increase in apparent affinity of Pt(NO2)4 2 block (Fig. 4), as did S1141K (8). Login to comment
151 The cartoon model of Fig. 1 suggests that it is proximity to the endogenous positive charge at K95, at least in terms of location along the axis of the pore, that determines the ability of introduced positive charges to strengthen Pt(NO2)4 2 block, since I344K and V345K (Fig. 4), together with S1141K (8), give the most potent block. Login to comment

PMID: 24671572 [PubMed] Linsdell P et al: "State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore."

No. Sentence Comment

31 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

PMID: 25673337 [PubMed] Rubaiy HN et al: "Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

79 e Mean KD values for Au(CN)2 - block for these channel constructs, as well as the additional positive charge mutants V345K/ E1371Q and S1141K/E1371Q, obtained as described in Fig. 1. f Relationship between the observed KD values for Au(CN)2 - block (at -100 mV) and the expected number of fixed positive charges in the pore inner vestibule in different channel constructs. Login to comment
90 Consistent with this, mutation of any of these three residues to lysine (I344K, V345K, S1141K) led to a significant strengthening of Au(CN)2 - block (Fig. 3d-f), with mean KD values at -100 mV being reduced by 18-fold (I344K/E1371Q), 17-fold (V345K/E1371Q) and 7-fold (S1141K/E1371Q), respectively. Login to comment