ABCMdb

ABCC7 p.Lys95Ser

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] Regulation of conductance by the number of fixed p... J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8. Zhou JJ, Li MS, Qi J, Linsdell P
Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore.
J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8., [PMID:20142516]

Abstract [show]
Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). 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. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. 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). 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(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a combination of failure to increase Cl(-) conductance and increased susceptibility to channel block by cytosolic substances.

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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
62 Fig. S1 shows the inhibitory effects of TLCS and lonidamine on wild type, K95S, K95S/S341K, and K95S/S1141K-CFTR. Login to comment
63 Fig. S2 shows single-channel recordings of K95S/S341K and S341K. Login to comment
74 Consistent with previous findings with other K95 mutants (Linsdell, 2005), removal of the positive charge at K95 in the K95S mutation is associated with significant reduction in the apparent potency of block by NPPB (Fig. 1, A and B). Login to comment
75 Woodhull analysis suggests that the apparent Kd (at 0 mV) is increased approximately sevenfold, from 12.4 ± 1.7 µM (n = 4) in wild type to 90.4 ± 17.6 µM (n = 4) in K95S (Fig. 1, B and D), without a significant change in apparent blocker voltage dependence (apparent valence, z, of 0.20 ± 0.02 [n = 4] in wild type and 0.16 ± 0.02 [n = 4] in K95S; P > 0.2). 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
100 This effect is clear in the K95S mutant, where unitary Cl currents appear close to the resolution for single-channel recording (Fig. 2 A). Login to comment
101 Although difficult to resolve unequivocally, the conductance of K95S channels appears to be <20% of wild-type conductance (Fig. 2, TA B L E I Major ATP species at each ATP concentration used Overall [ATP] [Mg2ATP0 ] [MgATP2 ] [HATP3 ] [NaATP3 ] [ATP4 ] 0.3 0.013 (4.2%) 0.25 (84.8%) 0.002 (0.8%) 0.021 (7.0%) 0.009 (3.0%) 1.0 0.027 (2.7%) 0.81 (81.4%) 0.011 (1.1%) 0.10 (10.2%) 0.044 (4.4%) 3.0 0.014 (0.5%) 1.68 (56.1%) 0.094 (3.1%) 0.84 (28.0%) 0.37 (12.2%) 10.0 0.003 (0.0%) 1.93 (19.3%) 0.56 (5.6%) 5.20 (52.0%) 2.30 (23.0%) For each overall ATP concentration given (in mM), the concentration (mM) and percentage of total ATP were calculated as described in Materials and methods. 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
109 Asterisks indicate a significant difference from K95S, and daggers indicate a significant difference from wild type (P < 0.05 in both cases). 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
120 Asterisks indicate a significant difference from K95S (P < 1010 ). 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
127 Thus, the S341K mutant was associated with very small unitary currents that were difficult to resolve unequivocally when introduced into either a wild-type or a K95S background (Fig. S2). 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
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
205 Also shown are the effects of 10 mM ATP on E1371Q () and K95S/S1141K/E1371Q () at 100 mV. 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
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
267 Thus, both S341K and K95S/S341K were associated with very low single-channel conductance (Fig. S2). Login to comment
289 The apparent valence of both TLCS and lonidamine was significantly reduced in K95S, although the apparent valence for NPPB was not significantly altered by this mutation. Login to comment
290 The meaning of the voltage dependence of the residual block observed in K95S is not clear, and because block is so weak in this mutant, we are reluctant to attach any physical meaning to the apparent valence. 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
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

[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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Sentences [show]
No. Sentence Comment
57 Similar results were previously reported for NPPB block of K95S/S341K and K95S/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

[hide] The CFTR ion channel: gating, regulation, and anio... Cold Spring Harb Perspect Med. 2013 Jan 1;3(1):a009498. doi: 10.1101/cshperspect.a009498. Hwang TC, Kirk KL
The CFTR ion channel: gating, regulation, and anion permeation.
Cold Spring Harb Perspect Med. 2013 Jan 1;3(1):a009498. doi: 10.1101/cshperspect.a009498., [PMID:23284076]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel with two remarkable distinctions. First, it is the only ATP-binding cassette (ABC) transporter that is known to be an ion channel--almost all others function as transport ATPases. Second, CFTR is the only ligand-gated channel that consumes its ligand (ATP) during the gating cycle--a consequence of its enzymatic activity as an ABC transporter. We discuss these special properties of CFTR in the context of its evolutionary history as an ABC transporter. Other topics include the mechanisms by which CFTR gating is regulated by phosphorylation of its unique regulatory domain and our current view of the CFTR permeation pathway (or pore). Understanding these basic operating principles of the CFTR channel is central to defining the mechanisms of action of prospective cystic fibrosis drugs and to the development of new, rational treatment strategies.

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Sentences [show]
No. Sentence Comment
218 Some K95 mutations (e.g., K95S) also markedly decrease singlechannelconductanceatbothdepolarizing and hyperpolarizing voltages (Zhou et al. 2010), which implies an effect on pore structure separate from a charge-attracting role for K95. Login to comment