ABCMdb

PMID: 20142516

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., [PubMed]

Sentences

No. Mutations Sentence Comment

16 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys 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 17 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys ABCC7 p.Ser1141Cys 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. Login to comment 19 ABCC7 p.Ser1141Lys 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). Login to comment 20 ABCC7 p.Ser1141Lys 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(NO2)4 2 in inside-out membrane patches. Login to comment 21 ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys Furthermore, in cell-attached patch recordings, apparent voltage- -dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Login to comment 27 ABCC7 p.Val510Ala Cys-less CFTR also included a mutation in the first nucleotide-binding domain (V510A) to increase protein expression in the cell membrane (Li et al., 2009). Login to comment 32 ABCC7 p.Glu1371Gln In some cases (see Fig. 7 and Fig. S4), macroscopic currents were recorded from cell-attached patches on unstimulated cells expressing spontaneously active E1371Q-CFTR channels. Login to comment 36 ABCC7 p.Glu1371Gln Because channels bearing the E1371Q mutation appeared to be spontaneously active even in the absence of ATP (see Fig. S4), inside-out patch recordings of this construct were made in the absence of PKA or ATP unless stated otherwise. Login to comment 37 ABCC7 p.Glu1371Gln Background current levels in these E1371Q mutants were determined at the end of the experiment by adding a high concentration (10 µM) of the specific CFTR inhibitor CFTRinh-172 (Ma et al., 2002) to the cytoplasmic solution in excised inside-out membrane patches. Login to comment 62 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys Fig. S1 shows the inhibitory effects of TLCS and lonidamine on wild type, K95S, K95S/S341K, and K95S/S1141K-CFTR. Login to comment 63 ABCC7 p.Lys95Ser ABCC7 p.Ser341Lys ABCC7 p.Ser341Lys Fig. S2 shows single-channel recordings of K95S/S341K and S341K. Login to comment 64 ABCC7 p.Ser1141Lys Fig. S3 shows the apparent time-and voltage-dependent inhibition of S1141K by intracellular ATP in inside-out patches. Login to comment 65 ABCC7 p.Glu1371Gln Fig. S4 compares the activity of wild type and E1371Q in on-cell and inside-out membrane patches. Login to comment 66 ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys Fig. S5 shows the block of wild type, S1141K, and S341K by intracellular NPPB. Login to comment 67 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Fig. S6 shows the block of E1371Q and E1371Q/S1141K by intracellular Pt(NO2)4 2 ions. Login to comment 74 ABCC7 p.Lys95Ser 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 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser 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 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys ABCC7 p.Ser341Lys ABCC7 p.Ser341Lys 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 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys 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 82 ABCC7 p.Glu1371Gln For E1371Q mutant channels, background currents were determined after the addition of 10 µM CFTRinh-172 (see above). Login to comment 87 ABCC7 p.Glu1371Gln Because of the effects of ATP on CFTR channel gating, these experiments were performed in an E1371Q background. Login to comment 98 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys 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 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys 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 ABCC7 p.Lys95Ser 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 ABCC7 p.Lys95Ser 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 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys , wild type (B); , K95S (B and C); , K95S/S341K (C); , K95S/S1141K (C). Login to comment 107 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys 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 ABCC7 p.Lys95Ser 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 111 ABCC7 p.Ser1141Lys Interestingly, the S1141K mutation alone led to a small but significant reduction in unitary conductance compared with wild type, to 8.02 ± 0.08 pS restored conductance at hyperpolarized voltages to 6.28 ± 0.06 pS (n = 9), 75% of wild-type values, and at depolarized voltages to 5.37 ± 0.04 pS (n = 10), 67% of Figure 2. Single-channel conductance is restored by moving a positive charge from TM1 to TM12. Login to comment 116 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys 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 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys (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 ABCC7 p.Lys95Ser Asterisks indicate a significant difference from K95S (P < 1010 ). Login to comment 121 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys 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 ABCC7 p.Lys95Ser ABCC7 p.Ser341Lys 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 131 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys ABCC7 p.Ser1141Cys (B) Mean fractional current remaining after the addition of CuPhe as a function of voltage in wild type (), K95C (), S1141C (), and K95C/S1141C (). Login to comment 132 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys ABCC7 p.Ser1141Cys Data values for K95C/S1141C were significantly different from wild type, K95C, or S1141C (P < 0.05 in each case) at all voltages examined. Login to comment 133 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys (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). Login to comment 135 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys (D) Example leak-subtracted macroscopic I-V relationships for cys-less K95C/S1141C-CFTR recorded under the same conditions as in A. Login to comment 139 ABCC7 p.Ser1141Cys In an interesting contrast to these effects, K95C/S1141C currents were not significantly affected by the addition of 300 µM Cd2+ ions (not depicted). Login to comment 144 ABCC7 p.Lys95Cys ABCC7 p.Ser341Cys ABCC7 p.Ser1141Cys In contrast, each of the mutants, K95C, S341C, and S1141C (all in a cys-less background), was strongly sensitive to both MTSES and MTSET. Login to comment 146 ABCC7 p.Lys95Cys ABCC7 p.Ser341Cys ABCC7 p.Ser1141Cys In contrast, MTSET inhibited currents carried by cys-less S341C and cys-less S1141C, but potentiated currents carried by cys-less K95C. Login to comment 147 ABCC7 p.Ser341Cys ABCC7 p.Ser1141Cys In S341C and S1141C, modification by the bulky MTSET molecule may partly occlude the pore, reducing Cl current. Login to comment 148 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys In contrast, in K95C, deposition of positive charge by reaction with MTSET may replace the function of the positive charge at this site that is lost as a consequence of the K95C mutation. Login to comment 149 ABCC7 p.Lys95Cys Consistent with this idea, MTSET modification converts the cys-less K95C I-V relationship from outwardly rectified (before modification) to linear or mildly inwardly rectified after modification (Fig. 4 B). Login to comment 152 ABCC7 p.Lys95Cys ABCC7 p.Ser341Cys ABCC7 p.Ser1141Cys 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. Login to comment 154 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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). Login to comment 155 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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. Login to comment 156 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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. Login to comment 158 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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. Login to comment 159 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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. Login to comment 161 ABCC7 p.Lys95Cys ABCC7 p.Ser341Cys A K95C/S341C double mutant did not yield functional currents in inside-out patches either without or after treatment with 5 mM DTT. Login to comment 162 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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. Login to comment 164 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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. Login to comment 165 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys On average, the application of CuPhe reduced current amplitude in cys-less K95C/S1141C by 84.7 ± 5.2% at +80 mV (n = 5). Login to comment 167 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Cys 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). Login to comment 168 ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys With a low extracellular Cl concentration, macroscopic currents in S1141K showed outward rectification leading to a flattening of the I-V relationship at hyperpolarized voltages (Fig. 5, A and B). Login to comment 170 ABCC7 p.Ser1141Lys We were therefore interested to know how the presence of two adjacent positive charges in this region of the inner vestibule-as presumably exist in the S1141K single mutant-would influence interactions with open-channel blockers. Login to comment 178 ABCC7 p.Ser1141Lys Replacing the anionic TES buffer with the cationic pH buffer Tris did not alter the unusual shape of the I-V relationship in S1141K (not depicted). Login to comment 179 ABCC7 p.Ser1141Lys In contrast, altering the ATP concentration did lead to striking time-and voltage-dependent changes in current amplitude that suggest an inhibitory effect of ATP on S1141K (Fig. S3). Login to comment 183 ABCC7 p.Ser1141Lys As a result, although PPi stimulated macroscopic current amplitude in S1141K at depolarized voltages, it actually inhibited current at the most hyperpolarized voltages studied (Fig. 5 D). Login to comment 184 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys 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 185 ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys Because the S1141K mutant did not strongly affect either unitary conductance or the linearity of the I-V relationship under symmetrical high Cl conditions (Fig. 2), we considered that the unusual I-V shape shown in Fig. 5 might reflect voltage-dependent block of S1141K by some negatively charged substance present in our intracellular solutions. Login to comment 186 ABCC7 p.Ser1141Lys However, the solutions Figure 5. Apparent inhibition of S1141K at hyperpolarized voltages. Login to comment 189 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys (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 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys 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 192 ABCC7 p.Ser1141Lys Asterisk indicates the voltage range over which the effects of PPi on wild type and S1141K were significantly different (P < 0.05). Login to comment 193 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Mean of data from four to five patches in B-D. tion of 10 mM ATP caused a potent inhibition of S1141K/E1371Q current during 400-ms hyperpolarizing voltage steps from a holding potential of +60 mV (Fig. 6 A). Login to comment 194 ABCC7 p.Glu1371Gln Time-dependent inhibition by ATP was still apparent; however, use of the E1371Q background provides direct evidence that ATP inhibits current flow because the amplitude of the current is greatly reduced. Login to comment 197 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys In contrast to the strong inhibition of S1141K/ E1371Q current by ATP under these conditions, 10 mM prolongation of CFTR channel open times (Vergani et al., 2003; Gadsby et al., 2006; Stratford et al., 2007). Login to comment 198 ABCC7 p.Glu1371Gln To our surprise, we found that the E1371Q mutant was constitutively active in on-cell recordings from intact BHK cells, and that even after excision into nominally ATP-free solutions, channel activity remained maximal (Fig. S4). Login to comment 199 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Although the reasons for this constitutive activity, which contrasts with a complete lack of spontaneous activity we observe for other CFTR constructs expressed in BHK cells, are unknown, it did allow us to quantify ATP effects on S1141K current amplitude in an E1371Q background in inside-out membrane patches, beginning with 0 ATP control conditions (Fig. 6). Login to comment 200 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys With a low extracellular Cl concentration (4 mM), the addi- Figure 6. Inhibition of S1141K/E1371Q-CFTR by intracellular ATP. Login to comment 201 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys (A) Example macroscopic currents carried by S1141K/E1371Q during hyperpolarizing voltage steps to between +60 and 100 mV recorded under conditions of low extracellular Cl concentration (4 mM). Login to comment 205 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys Also shown are the effects of 10 mM ATP on E1371Q () and K95S/S1141K/E1371Q () at 100 mV. Login to comment 206 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys (C) Example macroscopic S1141K/E1371Q currents during voltage steps to between +60 and 100 mV recorded with a high extracellular Cl concentration (154 mM) before (control) and after the addition of 10 mM Na2ATP to the intracellular solution in the absence of PKA. Login to comment 213 ABCC7 p.Glu1371Gln We took advantage of constitutive activity of E1371Q in BHK cells (see above) to monitor channel block in intact cells. CFTR activity was monitored using a voltage step protocol, both during on-cell recording and after patch excision into the inside-out configuration in the absence of ATP and PKA (Fig. 7). Login to comment 216 ABCC7 p.Glu1371Gln With high Cl concentration pipette solution, E1371Q showed outward rectification of the macroscopic I-V relationship during cell-attached recording (Fig. 7, A and B). Login to comment 218 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Outward rectification in cell-attached patches under these conditions was even more pronounced in S1141K/ E1371Q, and again this rectification was relieved by excision of the membrane patch, resulting in a linear I-V relationship in inside-out patches. Login to comment 221 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Such analysis revealed that current inhibition in cell-attached patches was significantly stronger in S1141K/E1371Q than in E1371Q at hyperpolarized voltages, both at high extracellular Cl concentrations (Fig. 7 C) and at low Cl concentrations (Fig. 7 D). Login to comment 224 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys 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 225 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Interestingly, the inhibition of S1141K/E1371Q by intracellular ATP was very much weaker when using a high extracellular Cl concentration (154 mM; Fig. 6 C) during voltage steps of the same duration. Login to comment 232 ABCC7 p.Ser1141Lys The apparent voltage-dependent block of S1141K by ATP, and likely also by PPi (Fig. 5), complicated studies of the interaction of this mutant with open-channel blockers like those studied in Fig. 1 and Fig. S1. Login to comment 233 ABCC7 p.Ser1141Lys Nevertheless, we were able to study NPPB block of S1141K under conditions where ATP block was weak (by using symmetrical Cl conditions) and in the absence of PPi. Login to comment 234 ABCC7 p.Ser1141Lys Under these conditions, a low concentration of NPPB (10 µM) had significantly more potent blocking effects on S1141K than on wild type (Fig. S5). Login to comment 235 ABCC7 p.Ser1141Lys The novel apparent inhibitory effects of ATP and PPi seen in S1141K but not in wild type suggest that this mutant might be particularly susceptible to block by polyvalent anions. Login to comment 237 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Under ATP-free conditions, block by intracellular Pt(NO2)4 2 was dramatically more potent in E1371Q/S1141K compared with E1371Q alone, leading to a 38-fold decrease in mean Kd(0) (Fig. S6). Login to comment 238 ABCC7 p.Ser1141Lys This result is consistent with the S1141K mutation preferentially increasing the strength of interactions between polyvalent anions and the pore. Login to comment 239 ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys Adding an extra positive charge to the inner vestibule of the pore decreases channel current in intact cells Because S1141K shows increased sensitivity to inhibition by intracellular anions such as ATP (Fig. 6), PPi (Fig. 5), NPPB (Fig. S5), and Pt(NO2)4 2 (Fig. S6), we wondered if this sensitivity would result in inhibition of channel currents in intact cells. CFTR channel currents are known to be subject to voltage-dependent inhibition by unknown anions present in the cytosol (Tabcharani Figure 7. Enhanced voltage-dependent inhibition in cell-attached patches in S1141K-CFTR. Login to comment 240 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys (A) Example macroscopic currents carried by E1371Q and S1141K/E1371Q-CFTR in cell-attached patches (left panels) after excision into the inside-out patch configuration (middle panels) and after the addition of 10 µM CFTRinh-172 to the intracellular solution (right panels). Login to comment 248 ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys 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 259 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys 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 260 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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). Login to comment 263 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys 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 ABCC7 p.Lys95Ser ABCC7 p.Ser341Lys ABCC7 p.Ser341Lys Thus, both S341K and K95S/S341K were associated with very low single-channel conductance (Fig. S2). Login to comment 268 ABCC7 p.Ser341Ala This is consistent with previous work, for example with S341A (McDonough et al., 1994), showing that mutations at this position are associated with dramatic loss of Cl conductance. Login to comment 271 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys This would not be surprising in evolutionary terms because maximization of Cl conductance is the physiologically meaningful role of the positive charge associated with K95 in this part of the inner vestibule of the pore, with interaction with channel blockers current amplitude in cell-attached patches as a fraction of current in the same patch after excision into the inside-out patch configuration for both E1371Q () and S1141K/E1371Q (), with 154 mM Cl (C) or 4 mM Cl (D) in the extracellular solution. Login to comment 275 ABCC7 p.Ser1141Lys This first became apparent to us as a novel sensitivity to block by intracellular ATP molecules (Fig. 6), although it appears that S1141K also increases sensitivity to PPi (Fig. 5), NPPB (Fig. S5), and Pt(NO2)4 2 (Fig. S6). Login to comment 276 ABCC7 p.Ser1141Lys Block by intracellular ATP appears as a voltage- and extracellular [Cl ]-dependent phenomenon (Fig. 6), suggesting that negatively charged ATP molecules are attracted into the pore of S1141K-CFTR by the increased number of fixed positive charges in the inner vestibule of this mutant. Login to comment 282 ABCC7 p.Ser1141Lys Given our uncertainty concerning which species of ATP are responsible for block of S1141K, it is difficult to draw conclusions on the relative effect of an additional fixed positive charge at this position on interactions with monovalent versus polyvalent anions. Login to comment 284 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Consistent with this, the apparent affinity of block by small, divalent Pt(NO2)4 2 anions was increased 38-fold in S1141K/ E1371Q compared with E1371Q (Fig. S6). Login to comment 285 ABCC7 p.Ser1141Lys In contrast, S1141K had a more modest effect on block by the larger, monovalent organic anion NPPB (Fig. S5), with the apparent Kd for this blocker being increased less than fourfold under different experimental conditions. Login to comment 286 ABCC7 p.Ser1141Lys Based on this highly limited survey of blocking anions, it seems reasonable to suggest that the additional positive charge present in the pore of S1141K channels favors interactions with polyvalent anions. An analogous situation has been described in K+ channel pores, where fixed negative charges in the channel protein preferentially attract polyvalent cationic blockers into the pore compared with monovalent K+ ions, leading to channel block and current rectification (Zhang et al., 2006). Login to comment 289 ABCC7 p.Lys95Ser 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 ABCC7 p.Lys95Ser 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 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys 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 294 ABCC7 p.Ser1141Lys The apparent functional interchangeability and physical proximity of K95 and S1141 allowed us to explore the consequences of increasing the number of positive charges in this region of the inner vestibule of the pore apparently available to interact with cytoplasmic anions from one (as we assume to exist in wild type) to two (in the S1141K mutant). Login to comment 295 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser1141Lys 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 298 ABCC7 p.Ser1141Lys Although S1141K was no better than wild type in terms of Cl conductance, it did show an increased We thank Dr. David Gadsby for providing the cys-less CFTR cDNA. Login to comment 338 ABCC7 p.Glu1371Gln As shown in Fig. 7, this inhibition is pronounced in BHK cells, leading to an 60% block of wild-type (actually E1371Q) currents in intact cells at 100 mV with high extracellular Cl . Login to comment 340 ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys Although block appears strong in wild type (E1371Q), it is still significantly strengthened in S1141K (Fig. 7), suggesting that the number of fixed positive charges in the inner vestibule of the pore controls interactions with endogenous cytoplasmic-blocking molecules, and therefore (in intact cells) overall channel function in terms of the rate of anion efflux. Login to comment 343 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1141Lys This may be because the presence of an additional positive charge favors attraction of polyvalent anions into the pore, whereas the normal substrates of CFTR-mediated transport (Cl and HCO3  ) are monovalent anions. An overall decrease in channel function in intact cells is demonstrated in on-cell current recordings (Fig. 7), which show decreased Cl currents (relative to unblocked currents after patch excision to the inside-out configuration) at hyperpolarized voltages in S1141K/E1371Q relative to E1371Q alone. Login to comment