ABCMdb

ABCC7 p.Lys95Gln

Predicted by SNAP2:	A: D (75%), C: D (75%), D: D (91%), E: D (85%), F: D (85%), G: D (85%), H: D (53%), I: D (80%), L: D (80%), M: D (75%), N: D (80%), P: D (91%), Q: D (75%), R: N (66%), S: D (63%), T: D (80%), V: D (80%), W: D (91%), Y: D (71%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: D, H: N, I: D, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D,

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[hide] Direct comparison of the functional roles played b... J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25. Ge N, Muise CN, Gong X, Linsdell P
Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25., 2004-12-31 [PMID:15504721]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, little is known about the relative functional contribution of different TM regions to the pore. We have used patch clamp recording to investigate the functional consequences of point mutations throughout the six transmembrane regions in the N-terminal part of the CFTR protein (TM1-TM6). A range of specific functional assays compared the single channel conductance, anion binding, and anion selectivity properties of different channel variants. Overall, our results suggest that TM1 and -6 play dominant roles in forming the channel pore and determining its functional properties, with TM5 perhaps playing a lesser role. In contrast, TM2, -3, and -4 appear to play only minor supporting roles. These results define transmembrane regions 1 and 6 as major contributors to the CFTR channel pore and have strong implications for emerging structural models of CFTR and related ATP-binding cassette proteins.

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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

[hide] Location of a common inhibitor binding site in the... J Biol Chem. 2005 Mar 11;280(10):8945-50. Epub 2005 Jan 5. Linsdell P
Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2005 Mar 11;280(10):8945-50. Epub 2005 Jan 5., 2005-03-11 [PMID:15634668]

Abstract [show]
Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a broad range of organic anions that enter the channel pore from its cytoplasmic end, physically occluding the Cl- permeation pathway. These open channel blocker molecules are presumed to bind within a relatively wide pore inner vestibule that shows little discrimination between different large anions. The present study uses patch clamp recording to identify a pore-lining lysine residue, Lys-95, that acts to attract large blocker molecules into this inner vestibule. Mutations that remove the fixed positive charge associated with this amino acid residue dramatically weaken the blocking effects of five structurally unrelated open channel blockers (glibenclamide, 4,4'-dinitrostilbene-2,2'-disulfonic acid, lonidamine, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and taurolithocholate-3-sulfate) when applied to the cytoplasmic face of the membrane. Mutagenesis of Lys-95 also induced amino acid side chain charge-dependent rectification of the macroscopic current-voltage relationship, consistent with the fixed positive charge on this residue normally acting to attract Cl- ions from the intracellular solution into the pore. These results identify Lys-95 as playing an important role in attracting permeant anions into the channel pore inner vestibule, probably by an electrostatic mechanism. This same electrostatic attraction mechanism also acts to attract larger anionic molecules into the relatively wide inner vestibule, where these substances bind to block Cl- permeation. Thus, structurally diverse open channel blockers of CFTR appear to share a common molecular mechanism of action that involves interaction with a positively charged amino acid side chain located in the inner vestibule of the pore.

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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

[hide] Identification of a second blocker binding site at... Mol Pharmacol. 2007 May;71(5):1360-8. Epub 2007 Feb 9. St Aubin CN, Zhou JJ, Linsdell P
Identification of a second blocker binding site at the cytoplasmic mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
Mol Pharmacol. 2007 May;71(5):1360-8. Epub 2007 Feb 9., [PMID:17293558]

Abstract [show]
Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is inhibited by a broad range of substances that bind within a wide inner vestibule in the pore and physically occlude Cl(-) permeation. Binding of many of these so-called open-channel blockers involves electrostatic interactions with a positively charged lysine residue (Lys95) located in the pore. Here, we use site-directed mutagenesis to identify a second blocker binding site located at the cytoplasmic mouth of the pore. Mutagenesis of a positively charged arginine at the cytoplasmic mouth of the pore, Arg303, leads to significant weakening of the blocking effects of suramin, a large negatively charged organic molecule. Apparent suramin affinity is correlated with the side chain charge at this position, consistent with an electrostatic interaction. In contrast, block by suramin is unaffected by mutagenesis of Lys95, suggesting that it does not approach close to this important pore-forming lysine residue. We propose that the CFTR pore inner vestibule contains two distinct blocker binding sites. Relatively small organic anions enter deeply into the pore to interact with Lys95, causing an open-channel block that is sensitive to both the membrane potential and the extracellular Cl(-) concentration. Larger anionic molecules can become lodged in the cytoplasmic mouth of the pore where they interact with Arg303, causing a distinct type of open-channel block that is insensitive to membrane potential or extracellular Cl(-) ions. The pore may narrow significantly between the locations of these two blocker binding sites.

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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

[hide] Direct and indirect effects of mutations at the ou... J Membr Biol. 2007 Apr;216(2-3):129-42. Epub 2007 Aug 3. Zhou JJ, Fatehi M, Linsdell P
Direct and indirect effects of mutations at the outer mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Membr Biol. 2007 Apr;216(2-3):129-42. Epub 2007 Aug 3., [PMID:17673962]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore is thought to contain multiple binding sites for permeant and impermeant anions. Here, we investigate the effects of mutation of different positively charged residues in the pore on current inhibition by impermeant Pt(NO(2)) (4) (2-) and suramin anions. We show that mutations that remove positive charges (K95, R303) influence interactions with intracellular, but not extracellular, Pt(NO(2))(4)(2-) ions, consistent with these residues being situated within the pore inner vestibule. In contrast, mutation of R334, supposedly located in the outer vestibule of the pore, affects block by both extracellular and intracellular Pt(NO(2))(4)(2-). Inhibition by extracellular Pt(NO(2))(4)(2-) requires a positive charge at position 334, consistent with a direct electrostatic interaction resulting in either open channel block or surface charge screening. In contrast, inhibition by intracellular Pt(NO(2))(4)(2-) is weakened in all R334-mutant forms of the channel studied, inconsistent with a direct interaction. Furthermore, mutation of R334 had similar effects on block by intracellular suramin, a large organic molecule that is apparently unable to enter deeply into the channel pore. Mutation of R334 altered interactions between intracellular Pt(NO(2))(4)(2-) and extracellular Cl(-) but not those between intracellular Pt(NO(2))(4)(2-) and extracellular Pt(NO(2))(4)(2-). We propose that while the positive charge of R334 interacts directly with extracellular anions, mutation of this residue also alters interactions with intracellular anions by an indirect mechanism, due to mutation-induced conformational changes in the protein that are propagated some distance from the site of the mutation in the outer mouth of the pore.

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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

[hide] Regulation of CFTR chloride channel macroscopic co... Am J Physiol Cell Physiol. 2011 Jan;300(1):C65-74. Epub 2010 Oct 6. Li MS, Holstead RG, Wang W, Linsdell P
Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate.
Am J Physiol Cell Physiol. 2011 Jan;300(1):C65-74. Epub 2010 Oct 6., [PMID:20926782]

Abstract [show]
The CFTR contributes to Cl and HCO transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl and HCO in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl and HCO regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO than when it contains Cl. This difference appears to reflect differences in the ability of extracellular HCO and Cl to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO concentrations and membrane potentials and can result in up to approximately 50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed.

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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

[hide] Alternating access to the transmembrane domain of ... J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1. Wang W, Linsdell P
Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).
J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1., [PMID:22303012]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

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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

[hide] Mechanism of direct bicarbonate transport by the C... J Cyst Fibros. 2009 Mar;8(2):115-21. Epub 2008 Nov 18. Tang L, Fatehi M, Linsdell P
Mechanism of direct bicarbonate transport by the CFTR anion channel.
J Cyst Fibros. 2009 Mar;8(2):115-21. Epub 2008 Nov 18., [PMID:19019741]

Abstract [show]
BACKGROUND: CFTR contributes to HCO(3)(-) transport in epithelial cells both directly (by HCO(3)(-) permeation through the channel) and indirectly (by regulating Cl(-)/HCO(3)(-) exchange proteins). While loss of HCO(3)(-) transport is highly relevant to cystic fibrosis, the relative importance of direct and indirect HCO(3)(-) transport it is currently unknown. METHODS: Patch clamp recordings from membrane patches excised from cells heterologously expressing wild type and mutant forms of human CFTR were used to isolate directly CFTR-mediated HCO(3)(-) transport and characterize its functional properties. RESULTS: The permeability of HCO(3)(-) was approximately 25% that of Cl(-) and was invariable under all ionic conditions studied. CFTR-mediated HCO(3)(-) currents were inhibited by open channel blockers DNDS, glibenclamide and suramin, and these inhibitions were affected by mutations within the channel pore. Cystic fibrosis mutations previously associated with disrupted cellular HCO(3)(-) transport did not affect direct HCO(3)(-) permeability. CONCLUSIONS: Cl(-) and HCO(3)(-) share a common transport pathway in CFTR, and selectivity between Cl(-) and HCO(3)(-) is independent of ionic conditions. The mechanism of transport is therefore effectively identical for both ions. We suggest that mutations in CFTR that cause cystic fibrosis by selectively disrupting HCO(3)(-) transport do not impair direct CFTR-mediated HCO(3)(-) transport, but may predominantly alter CFTR regulation of other HCO(3)(-) transport pathways.

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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

[hide] Molecular mechanism of arachidonic acid inhibition... Eur J Pharmacol. 2007 Jun 1;563(1-3):88-91. Epub 2007 Mar 3. Zhou JJ, Linsdell P
Molecular mechanism of arachidonic acid inhibition of the CFTR chloride channel.
Eur J Pharmacol. 2007 Jun 1;563(1-3):88-91. Epub 2007 Mar 3., [PMID:17397825]

Abstract [show]
Arachidonic acid inhibits the activity of a number of different Cl- channels, however its molecular mechanism of action is not known. Here we show that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by arachidonic acid is weakened following mutagenesis of two positively charged pore-lining amino acids. Charge-neutralizing mutants K95Q and R303Q both increased the Kd for inhibition from approximately 3.5 microM in wild type to approximately 17 microM. At both sites, the effects of mutagenesis were dependent of the charge of the substituted side chain. We suggest that arachidonic acid interacts electrostatically with positively charged amino acid side chains in the cytoplasmic vestibule of the CFTR channel pore to block Cl- permeation.

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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

[hide] Tuning of CFTR chloride channel function by locati... Biophys J. 2012 Oct 17;103(8):1719-26. doi: 10.1016/j.bpj.2012.09.020. Epub 2012 Oct 16. El Hiani Y, Linsdell P
Tuning of CFTR chloride channel function by location of positive charges within the pore.
Biophys J. 2012 Oct 17;103(8):1719-26. doi: 10.1016/j.bpj.2012.09.020. Epub 2012 Oct 16., [PMID:23083715]

Abstract [show]
High unitary Cl(-) conductance in the cystic fibrosis transmembrane conductance regulator Cl(-) channel requires a functionally unique, positively charged lysine residue (K95) in the inner vestibule of the channel pore. Here we used a mutagenic approach to investigate the ability of other sites in the pore to host this important positive charge. 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. Moving the positive charge also restored open-channel blocker interactions that are lost in K95Q. Introducing a second positive charge in addition to that at K95 did not increase conductance at any site, but did result in a striking increase in the strength of block by divalent Pt(NO(2))(4)(2-) ions. Based on the site dependence of these effects, we propose that although the exact location of the positive charge is not crucial for normal pore properties, transplanting this charge to other sites results in a diminution of its effectiveness that appears to depend on its location along the axis of the pore.

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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

[hide] State-dependent blocker interactions with the CFTR... Pflugers Arch. 2014 Dec;466(12):2243-55. doi: 10.1007/s00424-014-1501-7. Epub 2014 Mar 28. 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., [PMID:24671572]

Abstract [show]
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. 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. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

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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

[hide] Location of a permeant anion binding site in the c... J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12. Rubaiy HN, Linsdell P
Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12., [PMID:25673337]

Abstract [show]
In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognition and permeability in CFTR are discussed.

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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

[hide] Interactions between permeant and blocking anions ... Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17. 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., [PMID:25892339]

Abstract [show]
Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is antagonized by extracellular Cl(-). In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl(-) (and other anions) and cytoplasmic Pt(NO2)4(2-) ions inside the CFTR channel pore. 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. 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. 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. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl(-) and Pt(NO2)4(2-) to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl(-) binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl(-) permeation, are discussed.

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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