ABCMdb

ABCC7 p.Lys95Gln

None has been submitted yet.

Publications

PMID: 15504721 [PubMed] Ge N et al: "Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

44 Preliminary studies showed that some such mutations in TM1 did not yield expression of functional channels, and in these cases more conservative substitutions were employed (i.e. T94S, K95Q, A96V). Login to comment
76 However, the unitary conductance was drastically reduced by some mutations in TM1 (K95Q, Q98A, P99A) and TM6 (R334K, F337A) (Figs. 2-4). Login to comment
83 Interestingly, one mutant, K95Q, showed strong outward rectification (Figs. 5 and 6). Login to comment
102 The weakest block by Au(CN)2 - was observed in K95Q, T338A, R334K, and Q98A, consistent with these residues perhaps being associated with permeant anion binding sites inside the pore. Login to comment

PMID: 15634668 [PubMed] Linsdell P et al: "Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

74 A, example leak-subtracted current-voltage relationships for wild type CFTR and K95Q-CFTR following maximal channel activation with the protein kinase A catalytic subunit and PPi. In both cases, currents were recorded before (control) and after (ϩ glibenclamide) the addition of 30 ␮M glibenclamide to the intracellular solution. Login to comment
75 B, mean fraction of control current remaining (I/I0) after the addition of different concentrations of glibenclamide in wild type (E) and K95Q (●) at a membrane potential of -100 mV. Login to comment
76 Values are means of data from 3-5 patches fitted as described under "Experimental Procedures," with a Kd of 12.6 ␮M and an nH of 1.07 for wild type and a Kd of 107 ␮M for K95Q. Login to comment
77 Because the weak blocking effects of glibenclamide meant that a full concentration-inhibition curve could not be obtained for K95Q, nH was fixed at 1. Login to comment
78 C, voltage dependence of glibenclamide block in wild type (E) and K95Q (●). Login to comment
85 As shown in Fig. 2, inhibition of macroscopic CFTR current by glibenclamide, a potent intracellular open channel blocker, was greatly weakened in K95Q as compared with wild type. Login to comment
87 In K95Q, the Kd at this voltage was increased to 107 ␮M, an increase of ϳ8.5-fold. Login to comment
88 Furthermore, glibenclamide block of K95Q appeared only weakly dependent on voltage (Fig. 2C). Login to comment
91 These results, using a number of different amino acid substitutions of Lys-95, strongly suggest that side chain charge at this position is important in controlling the apparent affinity of glibenclamide block; the apparent Kd at -100 mV was not affected in the charge-conservative K95R but was significantly increased in charge-neutralizing mutants (K95A, K95C, K95Q) and most strongly increased in the charge-reversing K95E mutant. Login to comment
95 A, example leak-subtracted current-voltage relationships for wild type and K95Q-CFTR following maximal channel activation with the protein kinase A catalytic subunit and PPi. In each case, currents were recorded before (control) and after the addition of different CFTR channel blockers to the intracellular solution, namely 4,4Ј-dinitrostilbene-2,2Ј-disulfonic acid (DNDS) (100 ␮M), lonidamine (100 ␮M), NPPB (50 ␮M), or taurolithocholate-3-sulfate (TLCS) (50 ␮M). Login to comment
96 B, mean fraction of control current remaining (I/I0) after the addition of these concentrations of blockers in wild type (E) and K95Q (●) as a function of membrane potential. Login to comment
98 channel blockers on wild type and K95Q-CFTR are compared in Fig. 4. Login to comment
101 In contrast, at the same concentrations each of these substances had only weak inhibitory effects on K95Q-CFTR. Login to comment
103 Although this provides a rough approximation only, particularly as the estimated Kd was far greater than the concentration of blocker actually used in the case of K95Q, it does suggest that the blocking effects of each of these substances is in fact considerably more sensitive to mutagenesis of Lys-95 than are the blocking effects of glibenclamide. Login to comment
123 In contrast, the very low sensitivity of K95Q to other open channel blockers suggests that Lys-95 plays a dominant role in coordinating binding of these substances within the pore. Login to comment

PMID: 17293558 [PubMed] St Aubin CN et al: "Identification of a second blocker binding site at the cytoplasmic mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

92 Thus, the point mutation K95Q greatly weakened the blocking effects of glibenclamide, DNDS, lonid- amne, NPPB, and TLCS (Linsdell, 2005). Login to comment
105 A, example of leak-subtracted current-voltage relationships for K95Q and R303Q-CFTR recorded before (control) and after (ϩ suramin) the addition of 10 ␮M suramin to the intracellular solution. Login to comment
106 B, mean fraction of control current remaining (I/I0) after the addition of different concentrations of suramin at a membrane potential of -100 mV in K95Q (F) and R303Q (E). Login to comment
107 Mean of data from five to six patches is shown, fitted by eq. 1 (solid lines), giving Kd values of 11.6 ␮M for K95Q and 48.2 ␮M for R303Q. Login to comment
115 Figure 7 compares the blocking effects of intracellular glibenclamide in wild-type CFTR with the charge-neutralizing mutants R303Q and K95Q. Login to comment
116 It can be seen that glibenclamide inhibition is weakened in R303Q, although the effects of this mutant are not as dramatic as those seen in K95Q. Login to comment
117 Furthermore, a double mutant in which both of these positively charged residues were mutated to neutral glutamines (K95Q/R303Q) showed glibenclamide sensitivity similar to that of K95Q alone (Fig. 7). Login to comment
128 A, example of leak-subtracted current-voltage relationships for wild-type, R303Q, K95Q, and the K95Q/R303Q double mutant recorded before (control) and after (ϩ glibenclamide) the addition of 30 ␮M glibenclamide to the intracellular solution. Login to comment
163 Data for block of K95Q by DNDS, lonidamine, NPPB, and TLCS were taken from Linsdell (2005). Login to comment
164 Blocker K95Q R303Q Suramin 0 ϩϩ Glibenclamide ϩϩ ϩ DNDS ϩϩ ϩ Lonidamine ϩϩ 0 NPPB ϩϩ 0 TLCS ϩϩ 0 0, Ͻ2-fold change in Kd; ϩ, 2to 8-fold increase in Kd; ϩϩ, Ͼ8-fold increase in Kd. Login to comment
190 Whatever the mechanism, the far greater effects of the K95Q mutation on block by glibenclamide and DNDS relative to R303Q (Table 1) are consistent with the most important interaction underlying open-channel block by these two molecules being with Lys95. Login to comment

PMID: 17673962 [PubMed] Zhou JJ et al: "Direct and indirect effects of mutations at the outer mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

68 Whether studied under conditions of low (Fig. 1a) or high (Fig. 1b) extracellular ClÀ concentration, Pt(NO2)4 2À block is significantly weakened in both K95Q and R303Q (Figs. 1c, 2). Login to comment
70 Furthermore, a double mutant in which both of these positive charges are neutralized (K95Q/R303Q) generated currents that were almost completely insensitive to the blocking effects of Pt(NO2)4 2À (Fig. 1), consistent with the overall channel-blocking effects of Pt(NO2)4 2À ions being the result of interactions with both of these purportedly independent sites (St. Aubin et al., 2007). Login to comment
71 In spite of the weakened blocking effects of intracellular Pt(NO2)4 2À seen in both K95Q and R303Q, block of each of these two mutants was still significantly weakened by extracellular ClÀ ions (Figs. 1c, 2). Login to comment
74 However, unlike K95Q and R303Q, the R334Q mutant weakened Pt(NO2)4 2À block at low, but not high, extracellular ClÀ concentration (Fig. 2). Login to comment
76 As described above, weakened block by intracellular Pt(NO2)4 2À ions in K95Q and R303Q is consistent with Fig. 1 Removal of positive charges in the pore weakens block by intracellular Pt(NO2)4 2À ions. Login to comment
81 Mean of data from three to eight patches. Fitted lines are to equation 1, with the following parameters: wild type 4 mM external ClÀ , Kd(0) = 85.8 lM, zd = À0.201; wild type 154 mM external ClÀ , Kd(0) = 387 lM, zd = À0.344; K95Q 4 mM external ClÀ , Kd(0) = 403 lM, zd = À0.130; K95Q 154 mM external ClÀ , Kd(0) = 978 lM, zd = À0.227; R303Q 4 mM external ClÀ , Kd(0) = 300 lM, zd = À0.096; R303Q 154 mM external ClÀ , Kd(0) = 904 lM, zd = À0.197; R334Q 4 mM external ClÀ , Kd(0) = 286 lM, zd = À0.330; R334Q 154 mM external ClÀ , Kd(0) = 256 lM, zd = À0.307. Login to comment
82 Because block was so weak at this concentration, data for the K95Q/R303Q double mutant were not fitted current models of the pore that place these two positively charged amino acid residues within the pore inner vestibule. Login to comment
136 Example macroscopic current-voltage relationships for wild type and each R334 mutant studied, as well as mutations that neutralize positive charges involved in binding of intracellular Pt(NO2)4 2À (K95Q, R303Q), are compared in Figure 11a. Login to comment
140 Both K95Q and R303Q are associated with outward rectification under control conditions; this rectification was apparently weakened in the presence of extracellular Pt(NO2)4 2À , most likely due to voltage-dependent current inhibition, which was most prominent at depolarized voltages. Login to comment
141 The mean effects of 10 mM Pt(NO2)4 2À in each channel variant, analyzed as described above for wild type (Fig. 10c), are illustrated in Figure 11b (for R334 mutants) and 11c (for K95Q and R303Q). Login to comment
142 Considering only the data at +80 mV (Fig. 11d), where block of wild-type CFTR is strongest (Figs. 9, 10), the blocking effects of Pt(NO2)4 2À are slightly (but significantly) weakened in K95Q, R303Q and R334K (p < 0.05) but practically abolished in all other R334 mutants (p < 0.0005). Login to comment
143 In fact, only wild type, R334K, K95Q and R303Q - those mutants that retain a positive charge at position 334 - were significantly affected by 10 mM Pt(NO2)4 2À according to this analysis (as illustrated by the daggers in Fig. 11d, p < 0.001), whereas all mutants associated with removal of the positive charge at R334 showed no significant differences in the absence or presence of external Pt(NO2)4 2À (p > 0.15). Login to comment
149 Our present results suggest that block by intracellular Pt(NO2)4 2À involves interactions with positively charged amino acid side chains in the wide inner vestibule of the pore since mutations that remove these positive charges (K95Q, R303Q) lead to significant weakening of Pt(NO2)4 2À block (Figs. 1, 2). Login to comment
150 Furthermore, removal of both positive charges (in the K95Q/R303Q double mutant) gave rise to a channel that was almost completely resistant to the blocking effects of internal Pt(NO2)4 2À (Fig. 1), suggesting that both positive charges contribute to Pt(NO2)4 2À binding in the pore inner vestibule. Login to comment
178 Thus, all R334 mutants studied disrupted the dependence of intracellular Pt(NO2)4 2À block on extracellular ClÀ ions (Fig. 5), whereas Pt(NO2)4 2À block of both K95Q and R303Q remained Cl-dependent (Figs. 1, 2). Login to comment
226 Note the change in current rectification induced by extracellular Pt(NO2)4 2À in wild type, R334K, K95Q and R303Q but not other R334 mutants. Login to comment
228 ), R334E (5), R334H (j), R334K (), R334L (h), R334Q (u); c wild type (d), K95Q (m), R303Q (Å). Login to comment

PMID: 20926782 [PubMed] Li MS et al: "Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate."

No. Sentence Comment

118 To investigate whether these positively charged residues influence the strength of channel block by cytosolic anions in intact cells, we neutralized these charges in an E1371Q background. Login to comment
119 Figure 6A shows macroscopic currents carried by K95Q/ E1371Q-, R303Q/E1371Q-, and K978Q/E1371Q-CFTR in Fig. 4. Login to comment
130 Comparison of the fractional current in cell-attached patches seen in these mutants with those for E1371Q under the same ionic conditions (Fig. 1) suggests that block of each of these mutants is weakened under cell-attached conditions, particularly at hyperpolarized voltages, where block is usually strongest (Fig. 6B). Login to comment
131 Interestingly, the effects of the K95Q and R303Q mutations did not appear additive, with the K95Q/R303Q/E1371Q mutant showing relief of block following patch excision similar to that shown by K95Q/E1371Q (Fig. 6). Login to comment
132 Although channels bearing the K95Q mutation did give smaller currents in cell-attached patches than those seen following patch excision, this effect appeared voltage-independent, in contrast to the clearly voltage-dependent inhibition of E1371Q (Fig. 6B). Login to comment
134 In fact, because of its low single-channel conductance (16) and weakened interactions with intracellular Cl- , as well as blocking anions (31), K95Q could, in theory, show increased susceptibility to block by uncharged substances. Login to comment
133 In fact, because of its low single-channel conductance (16) and weakened interactions with intracellular Cl- , as well as blocking anions (31), K95Q could, in theory, show increased susceptibility to block by uncharged substances. Login to comment

PMID: 22303012 [PubMed] Wang W et al: "Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)."

No. Sentence Comment

188 Previously, we suggested that such a gate is located in the region of Lys-95/Gln-98 (TM1), Ile-344/Val-345 (TM6), Met-1140/ Ser-1141 (TM12) (17, 18, 21, 22). Login to comment
213 On opening, the occlusion in the pore dilates, perhaps in the region of Lys-95/Gln-98 (TM1), Ile-344/Val-345 (TM6) (22). Login to comment
195 Previously, we suggested that such a gate is located in the region of Lys-95/Gln-98 (TM1), Ile-344/Val-345 (TM6), Met-1140/ Ser-1141 (TM12) (17, 18, 21, 22). Login to comment
220 On opening, the occlusion in the pore dilates, perhaps in the region of Lys-95/Gln-98 (TM1), Ile-344/Val-345 (TM6) (22). Login to comment

PMID: 19019741 [PubMed] Tang L et al: "Mechanism of direct bicarbonate transport by the CFTR anion channel."

No. Sentence Comment

94 Fig. 4 shows that these same charge-neutralizing mutations (K95Q, R303Q) also drastically reduce the ability of these inhibitors to block HCO3 - currents under the same ionic conditions used in Fig. 3 (50 mM HCO3 - intracellular, 50 mM gluconate extracellular). Login to comment
95 Removal of one important positive charge in the K95Q mutant resulted in only small HCO3 - currents that were often difficult to resolve, nevertheless, Fig. 4 indicates that this mutation dramatically reduced the ability of both DNDS and glibenclamide to block HCO3 - currents. Login to comment
140 Mutations in the pore decrease the blocker sensitivity of bicarbonate currents (A) Example leak-subtracted I-V relationship recorded under the same ionic conditions used in Fig. 3B in K95Q-CFTR (left, centre panels) or R303Q-CFTR (right panel). Login to comment

PMID: 17397825 [PubMed] Zhou JJ et al: "Molecular mechanism of arachidonic acid inhibition of the CFTR chloride channel."

No. Sentence Comment

2 Charge-neutralizing mutants K95Q and R303Q both increased the Kd for inhibition from ~3.5 bc;M in wild type to ~17 bc;M. Login to comment
35 As also shown in Fig. 1, inhibition by arachidonic acid is dramatically weakened by point mutations which remove important positive charges from the pore inner vestibule, K95Q and R303Q, leading to approximately a 5-fold increase in Kd in both cases. Login to comment
44 (A) Example leak-subtracted current-voltage relationships recorded from inside-out membrane patches for wild type, K95Q, and R303Q-CFTR. Login to comment
46 (B) Mean fraction of control current remaining (I/I0) after the addition of different concentrations of arachidonic acid at a membrane potential of -100 mV, for wild type (cf;), K95Q (cb;) and R303Q (bc;). Login to comment
48 In each case the fitted lines are to Eq. (1), giving a Kd of 3.5 bc;M and nH of 1.54 for wild type, Kd of 17.2 bc;M and nH of 0.77 for K95Q, and Kd of 16.5 bc;M and nH of 1.06 for R303Q. 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

2 The loss of conductance observed in the K95Q mutation was >50% rescued by substituting a lysine for each of five different pore-lining amino acids, suggesting that the exact location of the fixed positive charge is not crucial to support high conductance. Login to comment
3 Moving the positive charge also restored open-channel blocker interactions that are lost in K95Q. Login to comment
27 Lysine mutations were made in both wild-type (WT) and K95Q backgrounds, resulting in channel constructs that contained two or one positively charged lysine residues in the putative inner vestibule of the pore, respectively. Login to comment
42 This requirement is clear in K95Q, which reduces conductance to ~15% of WT (Fig. 2, A-C). Login to comment
43 After neutralization of this endogenous positive charge by the K95Q mutation, introduction of a positive charge at other sites (by mutagenesis to lysine) caused a significant increase in unitary conductance to between 51 5 1% (in K95Q/A349K; n &#bc; 10) and 77 5 1% (in K95Q/M348K; n &#bc; 12) of WT conductance (Fig. 2, A-C), suggesting that a positive charge located at other positions in the pore can effectively rescue the WT conductance phenotype. Login to comment
50 As a result, the effects of removing the native positive charge by introducing the K95Q mutation appear highly background specific (Fig. 2 D), i.e., the effects of the K95Q mutation on conductance are far more dramatic in a WT background than when an additional positive charge is present. Login to comment
54 As shown in Fig. 3, addition of 50 mM NPPB to the cytoplasmic side of inside-out patches caused strong inhibition of WT CFTR but had no noticeable effect on K95Q channels. Login to comment
55 Additional mutations in a K95Q background to transplant the positive charge to pore-lining positions in TM1 (Q98K) or TM6 (I344K, V345K, M348K, and A349K) partially restored NPPB block (Fig. 3), although in no case was the block as strong as for the WT. Login to comment
56 The rank order of the apparent strength of NPPB block was WT > K95Q/V345K > K95Q/I344K > K95Q/Q98K ~ K95Q/ M348K ~ K95Q/A349K (Fig. 3 B). Login to comment
64 Note that the reduced single-channel current amplitude of K95Q is rescued by concurrent mutagenesis of other amino acids (Q98, I344, V345, M348, and A349) to lysine. Login to comment
66 The leftmost panel shows WT (green) and K95Q (red). Login to comment
67 The other five panels show the effects of the indicated lysine-introducing mutations in either a WT (open green symbols) or K95Q (open red symbols) background. Login to comment
68 In each case, the lines fitted to WT and K95Q data are indicated in green and red, respectively, as a reference. Login to comment
70 *Significant difference from WT (p < 0.05); # significant difference from K95Q (p < 1010 ); y significant difference from the same lysine mutation in a K95Q background (p < 0.05). Login to comment
72 In each case, the effect of the K95Q mutation was significantly reduced by the presence of an introduced lysine at other positions (*p < 1010 compared with WT). Login to comment
85 Transplanting the charge to other sites in TM1 (Q98) and TM6 (I344, V345, M348, and A349; Fig. 2), as well as S1141 in TM12 (8), results in restoration of at least 50% of the loss of conductance caused by the K95Q mutation. Login to comment
88 Although this residue does exert some influence over channel conductance (7), it presumably cannot compensate for the loss of positive charge in K95Q. Login to comment
96 *Significant difference from WT (p < 0.005); # significant difference from K95Q (p < 0.002). Login to comment
105 The weakening of blocker binding seen in K95Q is partially reversed by the second site mutations I344K and V345K, and to a lesser extent Q98K, M348K, and A349K. Login to comment
158 At its cytoplasmic entrance, the pore is wider (15,22), which may explain the weaker ability of the A349K mutation nearer the cytoplasmic end of the inner vestibule to restore single-channel conductance in a K95Q background (Fig. 2). 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

6 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
43 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 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
109 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
112 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
118 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
165 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 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
187 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

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

41 In contrast, the F337A mutation disrupts the normal Fig. 1 Block by intracellular Au(CN)2 - is weakened in K95Q/E1371Q channels. Example macroscopic IV relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of Au(CN)2 - to the intracellular (bath) solution at the concentrations stated. Login to comment
57 The macroscopic current reversal potential (VREV) Fig. 2 Block by intracellular SCN- and C(CN)3 - is weakened in K95Q/E1371Q channels. Example macroscopic IV relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of 10 mM SCN- to the intracellular (bath) solution. c Mean KD values for SCN- block for these channel constructs, obtained as described for Au(CN)2 - in Fig. 1. Login to comment
71 Thus, the mean KD was increased between 92-fold (at -100 mV) and 19-fold (at ?60 mV) in K95Q/E1371Q (Fig. 1d). Login to comment
72 Similar weakening of block in K95Q/E1371Q Fig. 3 Strength of Au(CN)2 - block is dependent on the number of positive charges in the pore inner vestibule. Login to comment
78 d Example macroscopic I-V relationships for I344K/E1371Q (left) and K95Q/I344K/E1371Q (right) CFTR channels recorded before (control) and after the addition of a low concentration (10 lM) of Au(CN)2 - to the intracellular (bath) solution. Login to comment
91 ''Moving`` the key positive charge from TM1 to TM6 by mutagenesis (in the K95Q/I344K/E1371Q mutant) resulted in Au(CN)2 - block that was intermediate in strength between E1371Q and I344K/E1371Q (Fig. 3e-f). Login to comment
94 a, b Example macroscopic I-V relationships for F337A/E1371Q CFTR channels recorded before (control) and after the addition of Au(CN)2 - (1 mM) or SCN- (10 mM) to the intracellular (bath) solution. c Mean KD values for Au(CN)2 - , SCN- , and C(CN)3 - (estimated at -100 mV as described in Figs. 1 and 2) compared in E1371Q, K95Q/ E1371Q, and F337A/E1371Q. Login to comment
98 As shown in Fig. 4, the F337A mutation had only a minor effect on binding of lyotropic Au(CN)2 - , SCN- and C(CN)3 - ions when compared to the K95Q mutation, suggesting that these anions can still bind relatively tightly in the pore even when lyotropic permeability selectivity is compromised. Login to comment
103 In contrast, neither K95Q nor I344K altered the permeability selectivity sequence; in fact, the only significant difference between either K95Q/ E1371Q or I344K/E1371Q compared to E1371Q was a small increase in PF/PCl in K95Q/E1371Q. Login to comment
109 Note that the normal lyotropic relationship between relative permeability and Gh is greatly reduced in F337A/E1371Q but retained in K95Q/E1371Q and I344K/E1371Q. Login to comment
112 Removal of a positive charge in the pore inner vestibule by mutagenesis (in the K95Q mutant) or by increasing pH (in K95H) dramatically increases the apparent KD (Figs. 1, 2, 3, 4), suggesting that this positive charge is required for tight binding of permeant anions. Login to comment
113 In fact, since Au(CN)2 - , SCN- and C(CN)3 - are permeant anions and presumably have access to the entire pore, it is possible that the residual block observed in K95Q and in K95H (at pH 9.0) reflects interactions with a different part of the pore. Login to comment
118 Thus, mutations that dramatically decrease (K95Q) or increase (I344 K) the strength of permeant anion binding inside the pore had only minimal effects on anion relative permeability (Fig. 5). Login to comment

PMID: 25892339 [PubMed] Linsdell P et al: "Interactions between permeant and blocking anions inside the CFTR chloride channel pore."

No. Sentence Comment

99 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 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 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
128 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 (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 There was no significant difference in these parameters for K95Q/E1371Q (E) (P N 0.05). Login to comment
140 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 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 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 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
162 (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
216 (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
222 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
234 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
282 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
311 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