ABCC7 p.Ser1141Cys
| Predicted by SNAP2: | A: N (53%), C: N (57%), D: D (66%), E: D (75%), F: D (71%), G: N (93%), H: D (75%), I: D (66%), K: D (80%), L: D (71%), M: D (59%), N: N (66%), P: D (75%), Q: D (53%), R: D (75%), T: D (53%), V: D (66%), W: D (80%), Y: D (75%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N, |
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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
17 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.
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ABCC7 p.Ser1141Cys 20142516:17:34
status: NEWX
ABCC7 p.Ser1141Cys 20142516:17:58
status: NEW131 (B) Mean fractional current remaining after the addition of CuPhe as a function of voltage in wild type (), K95C (), S1141C (), and K95C/S1141C ().
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ABCC7 p.Ser1141Cys 20142516:131:133
status: NEWX
ABCC7 p.Ser1141Cys 20142516:131:161
status: NEW132 Data values for K95C/S1141C were significantly different from wild type, K95C, or S1141C (P < 0.05 in each case) at all voltages examined.
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ABCC7 p.Ser1141Cys 20142516:132:21
status: NEWX
ABCC7 p.Ser1141Cys 20142516:132:82
status: NEW133 (C) Mean fractional current remaining after the addition of CuPhe at +80 mV for different channel variants as indicated, and for K95C/S1141C after washing with normal bath solution (wash) or with bath solution supplemented with 5 mM DTT (wash + DTT).
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ABCC7 p.Ser1141Cys 20142516:133:134
status: NEW135 (D) Example leak-subtracted macroscopic I-V relationships for cys-less K95C/S1141C-CFTR recorded under the same conditions as in A.
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ABCC7 p.Ser1141Cys 20142516:135:76
status: NEW139 In an interesting contrast to these effects, K95C/S1141C currents were not significantly affected by the addition of 300 µM Cd2+ ions (not depicted).
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ABCC7 p.Ser1141Cys 20142516:139:50
status: NEW144 In contrast, each of the mutants, K95C, S341C, and S1141C (all in a cys-less background), was strongly sensitive to both MTSES and MTSET.
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ABCC7 p.Ser1141Cys 20142516:144:51
status: NEW146 In contrast, MTSET inhibited currents carried by cys-less S341C and cys-less S1141C, but potentiated currents carried by cys-less K95C.
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ABCC7 p.Ser1141Cys 20142516:146:77
status: NEW147 In S341C and S1141C, modification by the bulky MTSET molecule may partly occlude the pore, reducing Cl current.
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ABCC7 p.Ser1141Cys 20142516:147:13
status: NEW152 The strong reactivity of cys-less K95C, S341C, and S1141C to intracellular MTSES and MTSET is consistent with the cysteine side chains introduced at these positions being exposed within the aqueous inner vestibule of the pore.
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ABCC7 p.Ser1141Cys 20142516:152:51
status: NEW154 The K95C/S1141C double mutant generated small macroscopic currents that showed outward rectification under symmetrical Cl concentration conditions (Fig. 3), as observed with all mutations that remove the charge at K95 (Linsdell, 2005), including K95C (Fig. 3).
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ABCC7 p.Ser1141Cys 20142516:154:9
status: NEW155 K95C/S1141C currents in inside-out patches were insensitive to the application of 5 mM of the reducing agent dithiothreitol (DTT; not depicted), suggesting that spontaneous disulfide bond formation between the two cysteine side chains is either negligible or without functional consequence.
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ABCC7 p.Ser1141Cys 20142516:155:5
status: NEW156 However, the oxidizing reagent CuPhe, which has been used to induce disulfide bond formation between introduced cysteines in other parts of the CFTR protein (Mense et al., 2006; Loo et al., 2008; Serohijos et al., 2008), led to a strong reduction in current amplitude in K95C/ S1141C (Fig. 3, A-C), suggesting a functional modification of the protein.
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ABCC7 p.Ser1141Cys 20142516:156:277
status: NEW158 Interestingly, neither wild-type CFTR currents nor the single mutants K95C or S1141C appeared sensitive to CuPhe under these conditions (Fig. 3, A-C), consistent with this agent acting by causing cross-linking of the two cysteine side chains introduced at these positions.
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ABCC7 p.Ser1141Cys 20142516:158:78
status: NEW159 Inhibition of K95C/S1141C by CuPhe was only partially reversed by washing; however, the degree of reversibility was significantly enhanced by the inclusion of 5 mM DTT in the wash solution (Fig. 3 C), consistent with CuPhe inhibition reflecting some oxidative process.
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ABCC7 p.Ser1141Cys 20142516:159:19
status: NEW162 Although these results suggest that K95C can be cross-linked to S1141C, they are potentially confounded by the presence of endogenous cysteine side chains in the CFTR protein.
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ABCC7 p.Ser1141Cys 20142516:162:64
status: NEW164 As shown in Fig. 3 D, cys-less K95C/S1141C also generated small, outwardly rectified currents in inside-out membrane patches that showed the same apparent sensitivity to CuPhe as that described above for these mutations in a wild-type background.
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ABCC7 p.Ser1141Cys 20142516:164:36
status: NEW165 On average, the application of CuPhe reduced current amplitude in cys-less K95C/S1141C by 84.7 ± 5.2% at +80 mV (n = 5).
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ABCC7 p.Ser1141Cys 20142516:165:80
status: NEW167 K95C/S1141C channel investigated the S1141K mutant at the macroscopic current level using depolarizing voltage ramp protocols like those used in Fig. 1, it became apparent that channel function had been altered in a way we had not anticipated from our initial single-channel experiments (see Fig. 2).
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ABCC7 p.Ser1141Cys 20142516:167:5
status: NEW260 Furthermore, we suggest that irreversible inhibition of channel function in the K95C/S1141C double mutant by the oxidizing agent CuPhe (Fig. 3) most likely reflects formation of a disulfide bridge between these two pore-lining cysteine side chains (Fig. 4).
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ABCC7 p.Ser1141Cys 20142516:260:85
status: NEW[hide] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]
Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.
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No. Sentence Comment
5 Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation.
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ABCC7 p.Ser1141Cys 21796338:5:5
status: NEW80 Application of MTSES (200 μM) or MTSET (2 mM) to the intracellular solution after channel activation with PKA, ATP, and PPi significantly altered macroscopic current amplitude in nine out of 19 cysteine-substituted mutants tested (N1138C, M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C, and S1149C; Figs. 1 and 2).
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ABCC7 p.Ser1141Cys 21796338:80:253
status: NEW82 In the central region of TM12 (N1138C, M1140C, and S1141C), macroscopic current amplitude was decreased by both MTSES and MTSET (Figs. 1 and 2).
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ABCC7 p.Ser1141Cys 21796338:82:51
status: NEW92 For MTS reagent-sensitive TM12 mutants located relatively deeply into the pore from its cytoplasmic end (N1138C, M1140C, S1141C, and T1142C), the rate of modification was estimated from the time course of macroscopic current amplitude change following application of MTSES (20-200 μM).
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ABCC7 p.Ser1141Cys 21796338:92:121
status: NEW93 As shown in Fig. 3a, modification was rapid in M1140C, S1141C, and T1142C, even using a low concentration of MTSES (20 μM), and noticeably slower in N1138C, even with 200 μM MTSES.
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ABCC7 p.Ser1141Cys 21796338:93:55
status: NEW96 To gain some information on the orientation of TM12 in the CFTR pore, we therefore examined whether the outermost cysteines introduced into this TM that were sensitive to internal MTS reagents (N1138C, M1140C, and S1141C) could also be modified by extracellular MTSET.
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ABCC7 p.Ser1141Cys 21796338:96:214
status: NEW101 As shown in Fig. 4a, patches excised from MTSET-pretreated cells expressing N1138C, M1140C, or S1141C all gave macroscopic currents that were decreased in amplitude following addition of 2 mM MTSET to the intracellular solution.
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ABCC7 p.Ser1141Cys 21796338:101:95
status: NEW103 These results suggest that none of N1138C, M1140C, or S1141C can be modified covalently by extracellular MTSET.
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ABCC7 p.Ser1141Cys 21796338:103:54
status: NEW106 We used a similar approach to determine whether N1138C, M1140C, S1141C, and T1142C, located relatively deeply into the pore from its cytoplasmic end and all strongly sensitive to inhibition by intracellular MTSES (Figs. 2 and 3), could be modified by MTSES pretreatment.
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ABCC7 p.Ser1141Cys 21796338:106:64
status: NEW118 Both S1141C and T1142C channels were again rendered insensitive to the test exposure to MTSES, again consistent with them having been covalently modified during pretreatment.
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ABCC7 p.Ser1141Cys 21796338:118:5
status: NEW120 These results, which are summarized quantitatively in Fig. 5d, suggest that while S1141C and T1142C can be modified by MTSES prior to channel activation, N1138C and M1140C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP.
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ABCC7 p.Ser1141Cys 21796338:120:82
status: NEW125 Extracellular MTSET did not appear able to modify the outermost of these internal MTS-sensitive cysteines (N1138C, M1140C, and S1141C; Fig. 4), consistent with MTS reagents not being permeant.
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ABCC7 p.Ser1141Cys 21796338:125:127
status: NEW140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9].
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ABCC7 p.Ser1141Cys 21796338:140:229
status: NEW143 a Example timecourses of macroscopic current amplitudes (measured at -50 mV) carried by N1138C, M1140C, S1141C, and T1142C as indicated in inside-out membrane patches.
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ABCC7 p.Ser1141Cys 21796338:143:104
status: NEW148 Using a similar approach, we find that in TM12, S1141C and T1142C can be readily modified by cytoplasmic MTSES prior to channel activation (Fig. 5), whereas N1138C and M1140C are modified rapidly after channel activation (Fig. 3) but very slowly, if at all, prior to channel activation (Fig. 5).
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ABCC7 p.Ser1141Cys 21796338:148:48
status: NEW151 Thus, a disulfide bridge can be formed between K95C in TM1 and S1141C in TM12, suggesting that the β carbon distance is in the range of ~5-8 Å for these two introduced cysteines [45].
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ABCC7 p.Ser1141Cys 21796338:151:63
status: NEW[hide] Structural basis for the channel function of a deg... J Gen Physiol. 2011 Nov;138(5):495-507. Bai Y, Li M, Hwang TC
Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).
J Gen Physiol. 2011 Nov;138(5):495-507., [PMID:22042986]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.
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No. Sentence Comment
114 (A) Current recorded at 50 mV in inside-out membrane patches containing thousands of cysless/S1141C-CFTR channels.
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ABCC7 p.Ser1141Cys 22042986:114:101
status: NEW133 Consistent with this idea, the S1141C-CFTR channel, when modified by 2-aminocarbonylethyl MTS (MTSACE), a similarly sized but neutral reagent, also displayed a smaller single-channel current amplitude (Fig. 3 D).
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ABCC7 p.Ser1141Cys 22042986:133:31
status: NEW137 (A and B) Measurements of the second-order reaction rate constant (MTSES ) of MTSES modification in the presence of 2 mM ATP for cysless/Q1144C(A)andcysless/S1141C (B).
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ABCC7 p.Ser1141Cys 22042986:137:182
status: NEW156 (C and D) Single-channel recordings show that the unitary current amplitude decreases after MTSET+ (C; blue) or MTSACE (D; green) exposure for cysless/S1141C.
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ABCC7 p.Ser1141Cys 22042986:156:151
status: NEW157 Linear fits to single-channel current measurements at different voltages yield unitary conductance values for cysless/S1141C of 7.0 pS before (C and D; black) and 6.1 pS after both MTSET+ (C; blue) and MTSACE (D; green) treatment.
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ABCC7 p.Ser1141Cys 22042986:157:118
status: NEW180 Note that the modification is faster in the presence of ATP than in the absence of ATP for cysless/N1148C-CFTR channels (B), whereas it is slower in the presence of ATP than in the absence of ATP for cysless/S1141C-CFTR channels (C).
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ABCC7 p.Ser1141Cys 22042986:180:208
status: NEW194 (A) Chemical modification of cysless/S1141C-CFTR channels by Texas red MTSEA+ (orange arrows) without ATP.
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ABCC7 p.Ser1141Cys 22042986:194:37
status: NEW198 (C) Second-order rate constants (MTSES ) of Texas red MTSEA+ modification for cysless/ S1141C-, cysless/N1148C-, cysless/ I344C-, and cysless/M348C-CFTR channels.
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ABCC7 p.Ser1141Cys 22042986:198:104
status: NEW[hide] Alignment of transmembrane regions in the cystic f... J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11. Wang W, El Hiani Y, Linsdell P
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11., [PMID:21746847]
Abstract [show]
Different transmembrane (TM) alpha helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.
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No. Sentence Comment
167 Also note that CuPhe had a stronger inhibitory effect on currents carried by K95C/I344C when measured at +80 mV compared with 80 mV; this same apparent voltage dependence was previously reported for K95C/S1141C under similar experimental conditions (Zhou et al., 2010).
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ABCC7 p.Ser1141Cys 21746847:167:212
status: NEW264 This kind of information is necessary to develop and validate three-dimensional structuralmodelsoftheporeregion.Previously,weshowed that a disulfide bond could be formed between K95C (in TM1) and S1141C (in TM12) (Zhou et al., 2010).
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ABCC7 p.Ser1141Cys 21746847:264:196
status: NEW[hide] Metal bridges illuminate transmembrane domain move... J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20. El Hiani Y, Linsdell P
Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel.
J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20., [PMID:25143385]
Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd(2+) bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane alpha-helices (TMs). Seven Cd(2+) bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd(2+) bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd(2+) bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd(2+) bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close.
Comments [show]
None has been submitted yet.
No. Sentence Comment
51 To investigate potential Cd2af9; bridges formed between pore-lining cysteine side chains exposed in the inner vestibule of the CFTR pore, we combined individual cysteines that we previously found to be accessible to cytoplasmically applied methanethiosulfonate reagents in three important pore-lining TMs: TM1 (K95C, Q98C) (13), TM6 (I344C, V345C, M348C, A349C) (15), and TM12 (M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C) (16), to generate a total of 50 double cysteine mutants (8 TM1:TM6; 14 TM1:TM12; 28 TM6:TM12).
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ABCC7 p.Ser1141Cys 25143385:51:389
status: NEW71 In contrast, the remaining seven double cysteine mutants, namely I344C/S1141C (Fig. 2, C and D), V345C/S1141C, M348C/ S1141C (Fig. 2, C and E), M348C/V1144C, M348C/W1145C, M348C/V1147C, and M348C/N1148C, all showed increased sensitivity to Cd2af9; , leading to a significant decrease in Ki as compared with either of the single cysteine mutants from which they were derived (estimated Ki values b0d; 50 òe;M; Fig. 3).
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ABCC7 p.Ser1141Cys 25143385:71:71
status: NEWX
ABCC7 p.Ser1141Cys 25143385:71:103
status: NEWX
ABCC7 p.Ser1141Cys 25143385:71:118
status: NEW77 In particular, M348C/S1141C was associated with a dramatic increase in sensitivity to Cd2af9; (Fig. 2, C and E, and Fig. 3) with an estimated Ki of 0.14 afe; 0.02 òe;M (n afd; 5).
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ABCC7 p.Ser1141Cys 25143385:77:21
status: NEW80 In each case, PPi treatment resulted in a weakening of Cd2af9; inhibition (Fig. 4A) and a significant increase in Ki (Fig. 4B) of between 2.3-fold (in I344C/S1141C) and 97-fold (in M348C/ S1141C).
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ABCC7 p.Ser1141Cys 25143385:80:160
status: NEWX
ABCC7 p.Ser1141Cys 25143385:80:191
status: NEW83 As shown in Fig. 5, all E1371Q-containing channels tested were only weakly sensitive to inhibition by Cd2af9; , resulting in a significant increase in Ki both in single cysteine (I344C, M348C, S1141C) and in double cysteine (I344C/S1141C, Fig. 5, A-C; M348C/S1141C, Fig. 5, A, D, and E) mutant channels.
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ABCC7 p.Ser1141Cys 25143385:83:196
status: NEWX
ABCC7 p.Ser1141Cys 25143385:83:234
status: NEWX
ABCC7 p.Ser1141Cys 25143385:83:261
status: NEW84 However, the effect of the E1371Q mutation was greater in the double cysteine mutants; this gating mutation increased Ki 30-fold in I344C/S1141C (Fig. 5C) and 2500-fold in M348C/S1141C (Fig. 5E).
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ABCC7 p.Ser1141Cys 25143385:84:138
status: NEWX
ABCC7 p.Ser1141Cys 25143385:84:178
status: NEW98 C, sample time courses (upper panels) and I-V curves (lower panels) recorded from similar experiments for the double cysteine mutants I344C/S1141C (left) and M348C/S1141C (right).
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ABCC7 p.Ser1141Cys 25143385:98:140
status: NEWX
ABCC7 p.Ser1141Cys 25143385:98:164
status: NEW114 However, one mutant, K95C/S1141C, was apparently stimulated by Cd2af9; (Fig. 6A), and this stimulation was of a greater magnitude than that seen in the K95C single cysteine mutant (Figs. 6C and 7).
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ABCC7 p.Ser1141Cys 25143385:114:26
status: NEW115 This suggests that a Cd2af9; bridge may form between K95C in TM1 and S1141C in TM12.
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ABCC7 p.Ser1141Cys 25143385:115:72
status: NEW116 As with K95C itself, the stimulatory effects of Cd2af9; on K95C/S1141C were abolished by the E1371Q mutation (Fig. 6C) or by treatment with PPi (data not shown), suggesting that the observed effects of Cd2af9; are due to an increase in channel open probability and a stabilization of the channel open state.
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ABCC7 p.Ser1141Cys 25143385:116:67
status: NEW127 Note that the Ki for M348C/S1141C (0.14 afe; 0.02 òe;M, n afd; 5) is too small to be visible on this scale, but was significantly different from either M348C or S1141C alone (p b0d; 0.0005; see also Fig. 2E).
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ABCC7 p.Ser1141Cys 25143385:127:27
status: NEWX
ABCC7 p.Ser1141Cys 25143385:127:171
status: NEW137 Thus, M348C is able to form Cd2af9; bridges with cysteines at multiple positions in TM12 (S1141C, Q1144C, W1145C, V1147C, N1148C) (Fig. 8B), and S1141C is able to form Cd2af9; bridges with cysteines both in TM1 (K95C) and in TM6 (I344C, V345C, M348C) (Fig. 8C).
X
ABCC7 p.Ser1141Cys 25143385:137:93
status: NEWX
ABCC7 p.Ser1141Cys 25143385:137:148
status: NEW140 Similarly, S1141C is able to form Cd2af9; bridges with different sites in TM6 (Fig. 8C).
X
ABCC7 p.Ser1141Cys 25143385:140:11
status: NEW146 Most striking in this respect was the M348C/S1141C mutant FIGURE 4.
X
ABCC7 p.Ser1141Cys 25143385:146:44
status: NEW148 A, mean fractional current remaining following the addition of different concentrations of Cd2af9; in M348C/S1141C channels in the presence of PKA and ATP (F) or following activation by PKA and ATP followed by treatment with 2 mM PPi to maximize channel open probability (E).
X
ABCC7 p.Ser1141Cys 25143385:148:111
status: NEW152 The unusually high apparent Cd2af9; binding affinity of this double cysteine mutant (Figs. 3B and 4B), more than 700-fold lower Ki than the corresponding single cysteine mutants (Figs. 2E and 3), suggests that M348C and S1141C are uniquely well positioned to coordinate tight Cd2af9; binding in closed channels.
X
ABCC7 p.Ser1141Cys 25143385:152:223
status: NEW153 However, when the channel is open, these two cysteines do not appear to coordinate Cd2af9; at all because the apparent Cd2af9; affinity in M348C/S1141C/ E1371Q channels appears the same as in M348C/E1371Q or S1141C/E1371Q (Fig. 5, D and E).
X
ABCC7 p.Ser1141Cys 25143385:153:151
status: NEWX
ABCC7 p.Ser1141Cys 25143385:153:214
status: NEW154 In contrast, a single identified Cd2af9; bridge between TMs 1 and 12 (K95C/S1141C) appears to stabilize the channel open state, leading to a significant increase in channel current (Fig. 6).
X
ABCC7 p.Ser1141Cys 25143385:154:78
status: NEW157 If we assume that the effects of Cd2af9; bridges on the stability of the channel open and closed states reflects changes in the relative positions of cysteine side chains introduced into different TMs in these states, then our results would suggest that a number of side chains in TMs 6 and 12 are in close proximity in closed channels, and K95C (TM1) and S1141C (TM12) are in close proximity in open channels.
X
ABCC7 p.Ser1141Cys 25143385:157:359
status: NEW164 A, sample time courses and I-V curves illustrating the low Cd2af9; sensitivity of constitutively active I344C/S1141C/E1371Q (left panels) and M348C/S1141C/E1371Q (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2.
X
ABCC7 p.Ser1141Cys 25143385:164:113
status: NEWX
ABCC7 p.Ser1141Cys 25143385:164:151
status: NEW175 As described above, several different Cd2af9; bridges can form between M348C (TM6) and TM12 (Fig. 8, A and B), as well as between S1141C (TM12) and TM6; all appear to stabilize the closed state (Figs. 3 and 4).
X
ABCC7 p.Ser1141Cys 25143385:175:133
status: NEW176 Thus, for the reasons described earlier, there may be FIGURE 6. Cadmium increases macroscopic current amplitude in K95C and K95C/S1141C channels in a gating-dependent manner.
X
ABCC7 p.Ser1141Cys 25143385:176:129
status: NEW177 A, sample time courses and I-V curves showing Cd2af9; concentration-dependent increases in macroscopic current amplitude in K95C (left panels) and K95C/S1141C (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2.
X
ABCC7 p.Ser1141Cys 25143385:177:155
status: NEW181 C, Cd2af9; causes a significantly greater increase in K95C/S1141C current amplitude as compared with K95C (p b0d; 0.0001); this increase is not observed in K95C/S1141C/E1371Q channels.
X
ABCC7 p.Ser1141Cys 25143385:181:62
status: NEWX
ABCC7 p.Ser1141Cys 25143385:181:167
status: NEW188 In contrast to this consistent pattern of Cd2af9; bridge formation between TMs 6 and 12 stabilizing the closed state, a single Cd2af9; bridge between K95C (TM1) and S1141C (TM12) appears to stabilize the open state (Fig. 6).
X
ABCC7 p.Ser1141Cys 25143385:188:171
status: NEW198 E, proposed graphic model for Cd2af9; bridge formation between the key sites illustrated in D in open and closed states, and proposed implications for conformationalrearrangementofthesethreeTMsassociatedwithchannelgating.TheclosedstateisstabilizedbyaCd2af9; bridgebetweenM348C(TM6)andS1141C (TM12), and the open state is stabilized by a Cd2af9; bridge between K95C (TM1) and S1141C (TM12).
X
ABCC7 p.Ser1141Cys 25143385:198:384
status: NEW