ABCMdb

ABCC7 p.Ser341Lys

ClinVar:	c.1021T>C , p.Ser341Pro D , Pathogenic
CF databases:	c.1021T>C , p.Ser341Pro D , CF-causing ; CFTR1: This homozygous mutation was identified in two sister siblings with CF.
Predicted by SNAP2:	A: D (71%), C: D (80%), D: D (91%), E: D (85%), F: D (85%), G: D (71%), H: D (85%), I: D (85%), K: D (85%), L: D (91%), M: D (85%), N: D (75%), P: D (91%), Q: D (80%), R: D (91%), T: D (53%), V: D (85%), W: D (85%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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Publications
[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
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
66 Fig. S5 shows the block of wild type, S1141K, and S341K by intracellular NPPB. 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
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
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
248 The ability of a positive charge located in TM1 (K95), TM6 (S341K), or TM12 (S1141K) to support blocker interactions is consistent with each of these TMs influencing the movement of blocking anions in the pore. Login to comment
267 Thus, both S341K and K95S/S341K were associated with very low single-channel conductance (Fig. S2). 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

[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
60 As shown in Fig. 4, block by Pt(NO2)4 2 was significantly strengthened in each of the mutants Q98K, I344K, V345K, M348K, and A349K, as well as in the previously unstudied S341K. Login to comment
61 At 0 mV membrane potential, the apparent Kd for Pt(NO2)4 2 block was in the rank order V345K (3.3 5 0.9 mM, n &#bc; 7) % I344K (4.5 5 0.7 mM, n &#bc; 6) < S341K (26.6 5 1.8 mM, n &#bc; 7) < M348K (80.9 5 7.2 mM, n &#bc; 5) % Q98K (95.4 5 11.0 mM, n &#bc; 6) % A349K (117.4 5 7.7 mM, FIGURE 2 Single-channel conductance is restored by moving the positive charge from K95. Login to comment
86 Only S341K, located more deeply in the pore from its cytoplasmic end, was unable to support high conductance (8). Login to comment
146 Again this appears to be a relatively nonsite-specific effect of positive charge, since all mutants studied (Q98K, S341K, I344K, V345K, M348K, and A349K) led to significant increase in apparent affinity of Pt(NO2)4 2 block (Fig. 4), as did S1141K (8). Login to comment
157 At the outermost extent of this inner pore region, the open-channel pore may become too narrow to accommodate a positive charge (as previously proposed to explain the low single-channel conductance of S341K and other mutations at the same site (8)). Login to comment