ABCMdb

PMID: 21746847

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., [PubMed]

Sentences

No. Mutations Sentence Comment

19 ABCC7 p.Lys95Cys Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Login to comment 22 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys 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. Login to comment 60 ABCC7 p.Val510Ala As in our recent study on CFTR-TM6 (El Hiani and Linsdell, 2010), we have used a human CFTR variant in which all 18 endogenous cysteine residues had been substituted by other amino acids (as described in Mense et al., 2006), and which also includes a mutation in the first nucleotide-binding domain (V510A) to increase protein cross-linked residues in a current homology model of the CFTR membrane-spanning domain. Login to comment 64 ABCC7 p.Val510Ala Using site-directed mutagenesis, we substituted cysteines for each of 21 consecutive amino acids, from Y84 near the putative intracellular end of TM1 to R104 near the extracellular end, in a V510A cys-less background (see Materials and methods). Login to comment 66 ABCC7 p.Glu92Cys The one exception was E92C, which failed to generate any measureable current, even after multiple attempts with two independently constructed mutant cDNAs. Login to comment 70 ABCC7 p.Arg104Cys This list of MTS reagent-insensitive mutants includes R104C (Figs. 1 B and 2), which we have previously shown to be sensitive to modification by externally applied MTSES and MTSET (Zhou et al., 2008). Login to comment 71 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys In contrast, macroscopic currents carried by four mutants, K95C, Q98C, P99C, and L102C, were found to be significantly and rapidly sensitive to the application of both MTSES and MTSET (Figs. 1-3). Login to comment 96 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Fig. S2 shows the lack of sensitivity to the reducing agent DTT of macroscopic currents carried by the double-cysteine mutant channels K95C/ I344C and Q98C/I344C. Login to comment 98 ABCC7 p.Lys95Cys ABCC7 p.Leu102Cys As shown in Fig. 3 A, MTSES modification was rapid in K95C, even when a low concentration of MTSES (20 µM) was used, and considerably slower in L102C (using 200 µM MTSES). Login to comment 102 ABCC7 p.Gln98Cys (A) Example time courses of macroscopic currents (measured at +50 mV) carried by cys-less CFTR and Q98C inside-out membrane patches. Login to comment 104 ABCC7 p.Gln98Cys Note that whereas these MTS reagents have no effect on cys-less CFTR current amplitude, they cause rapid inhibition (MTSES) or augmentation (MTSET) of current carried by Q98C. Login to comment 105 ABCC7 p.Lys95Cys ABCC7 p.Arg104Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys (B) Example leak-subtracted I-V relationships for cys-less CFTR, K95C, Q98C, P99C, L102C, and R104C, recorded from inside-out membrane patches after maximal channel activation with 20 nM PKA, 1 mM ATP, and 2 mM PPi. Login to comment 112 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys As shown in Fig. 4 A, patches excised from MTSET-pretreated cells expressing K95C, Q98C, P99C, or L102C all gave macroscopic currents that were increased in amplitude after the addition of 2 mM constants was that modification was faster for cysteines introduced closer to the intracellular end of TM1, and slower for cysteines located more deeply along the axis of TM1 (Fig. 3 B). Login to comment 118 ABCC7 p.Glu92Cys Note that no currents were recorded from patches excised from cells transfected with E92C cDNA (see Results). Login to comment 123 ABCC7 p.Lys95Cys ABCC7 p.Leu102Cys (A) Example time courses of macroscopic currents (measured at 50 mV during brief voltage excursions from a holding potential of 0 mV) carried by K95C (left) and L102C (right) as indicated, in inside-out membrane patches. Login to comment 125 ABCC7 p.Lys95Cys ABCC7 p.Leu102Cys In each case, MTSES (20 µM for K95C and 200 µM for L102C) was applied to the cytoplasmic face of the patch at time zero (as indicated by the hatched bar at the bottom of each panel). Login to comment 128 ABCC7 p.Lys95Cys Asterisks indicate a significant difference from MTSES modification of K95C (P < 0.005), and daggers indicate a significant difference from MTSES modification of the same mutant (P < 0.05). Login to comment 136 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Gln98Cys Whereas K95C channels were again rendered insensitive to a test exposure to MTSES, again consistent with them having been covalently modified during pretreatment, currents carried by Q98C, P99C, MTSET to the intracellular solution. Login to comment 138 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys These results suggest that none of K95C, Q98C, P99C, or L102C can be modified covalently by extracellular MTSET. Login to comment 139 ABCC7 p.Thr338Cys ABCC7 p.Met348Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys Our work concerning intracellular MTS reagent modification in TM6 also identified some cysteines that could be modified in both activated and nonactivated channels (e.g., V345C and M348C), and others that could apparently be modified only after channel activation (e.g., T338C, S341C, and I344C), suggesting a state-dependent conformational change that alters access of internally applied MTS reagents into the pore (El Hiani and Linsdell, 2010). Login to comment 141 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys We used a similar approach to determine if K95C, Q98C, P99C, and L102C could be modified by MTSES pretreatment. Login to comment 148 ABCC7 p.Lys95Cys ABCC7 p.Gln98Cys Proximity and alignment of TMs 1 and 6 The results described in Fig. 5, suggesting that K95C is accessible to cytoplasmic MTSES in nonactivated channels but that Q98C is accessible only in activated channels, imply that K95 and Q98 may lie close to the putative barrier within the pore that we recently proposed to regulate access from the cytoplasmic solution (El Hiani and Linsdell, 2010). Login to comment 149 ABCC7 p.Leu102Cys In TM6, I344 and V345 were proposed to lie on either side of this barrier, based on similar and L102C were all strongly inhibited by the application of the test dose of MTSES, suggesting that they were not covalently modified by MTSES pretreatment. Login to comment 150 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys These results, which are summarized quantitatively in Fig. 5 C, suggest that although K95C can be modified by MTSES before channel activation, Q98C, P99C, and L102C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP. Login to comment 162 ABCC7 p.Met348Cys ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys (A-C) Example leak-subtracted I-V relationships for K95C/I344C (A), Q98C/I344C (B), and Q98C/M348C (C) after channel activation with 20 nM PKA and 1 mM ATP. Login to comment 166 ABCC7 p.Met348Cys ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys Note that cys-less CFTR, the single mutants K95C, Q98C, or I344C, and the double mutant Q98C/M348C were all insensitive to CuPhe under these conditions. Login to comment 167 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ser1141Cys 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). Login to comment 168 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys In contrast, the inhibitory effects of CuPhe on Q98C/I344C were similar when measured at 80 mV or +80 mV. Login to comment 170 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys (E) Mean effects of CuPhe (black bars), CuPhe followed by washing with normal bath solution (white bars), and CuPhe followed by DTT (gray bars) on macroscopic current amplitude in K95C/I344C (left) and Q98C/I344C (right) at +80 mV. Login to comment 172 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Mean of data from three to seven patches is shown in D and E. both K95C/I344C and Q98C/I344C by CuPhe was not reversed by washing CuPhe from the bath; however, partial reversal was seen when 5 mM DTT was applied in the continued presence of CuPhe (Fig. 6, A, B, and E), consistent with CuPhe inhibition of these channels reflecting some oxidative process. Login to comment 173 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys The results shown in Fig. 6 suggest that disulfide bond formation can occur between K95C and I344C and between Q98C and I344C after channel activation. Login to comment 175 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys After patch excision, inside-out patches from cells expressing either K95C/I344C or Q98C/ I344C were treated with cytoplasmic CuPhe for 2 min, after which CuPhe was washed from the bath and currents were activated using PKA and ATP, as usual. Login to comment 178 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Both K95C/I344C and Q98C/I344C channel currents were also potently inhibited by the addition of Cu2+ ions alone (without phenanthroline) to the bath (Fig. 8). Login to comment 180 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Each of the single mutants K95C, Q98C, and I344C showed reversible "paired" mutants with one cysteine introduced into each of TM1 (at K95 or Q98) and TM6 (at I344 and V345). Login to comment 181 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Val345Cys ABCC7 p.Gln98Cys Unfortunately, the double mutants K95C/V345C and Q98C/V345C did not yield functional currents when expressed in BHK cells, even after treatment with DTT to break any possible disulfide bonds; a similar lack of functional expression was previously reported for K95C/S341C (Zhou et al., 2010). Login to comment 182 ABCC7 p.Met348Cys ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys However, K95C/ I344C, Q98C/I344C, and Q98C/M348C did generate macroscopic PKA- and ATP-dependent currents in inside-out patches. Login to comment 184 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys However, the oxidizing agent CuPhe, which has previously 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; Zhou et al., 2010), led to a strong reduction in current amplitude in both K95C/I344C and Q98C/I344C (Fig. 6). Login to comment 185 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys Neither cys-less CFTR nor the single mutants K95C, Q98C, or I344C appeared sensitive to CuPhe under these conditions (Fig. 6 D), consistent with this agent acting by causing disulfide bond formation between the two introduced cysteine side chains in the double mutants K95C/I344C and Q98C/I344C. Login to comment 186 ABCC7 p.Met348Cys ABCC7 p.Gln98Cys Furthermore, the lack of effect of CuPhe on Q98C/M348C indicated that not all double-cysteine mutants were CuPhe sensitive, which we take to indicate that only nearby cysteine side chains can be cross-linked by this reagent. Login to comment 188 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys (A and B) Example leak-subtracted I-V relationships for K95C/I344C (A) and Q98C/I344C (B) after channel activation with 20 nM PKA and 1 mM ATP in inside-out patches that had been pretreated with CuPhe for 2 min, and then washed to remove CuPhe. Login to comment 199 ABCC7 p.Arg104Cys However, R104C was not sensitive to intracellularly applied MTS reagents (Figs. 1 and 2), consistent with the idea that such reagents are not able to permeate the CFTR pore (Alexander et al., 2009; El Hiani and Linsdell, 2010). Login to comment 200 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Our results concerning the accessibility of cysteines introduced into TM1 are summarized, and compared inhibition by Cu2+ that was of intermediate potency between the cys-less background and the double mutants K95C/I344C and Q98C/I344C. Login to comment 206 ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys (A) Example leak-subtracted I-V relationships for cys-less (left), I344C (center), and Q98C/I344C (right) after channel activation with 20 nM PKA and 1 mM ATP. Login to comment 208 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys In all channel constructs studied, these inhibitory effects of Cu2+ were readily and rapidly reversed by washing Cu2+ from the bath (for example, see right panel for complete reversal of the strong blocking effect on Q98C/I344C). Login to comment 209 ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys (B) Mean fractional current remaining after the addition of different concentrations of Cu2+ for cys-less (), I344C (), and Q98C/I344C (). Login to comment 210 ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Data are fitted as described in Materials and methods, giving Kd = 129 µM and nH = 1.36 for cys-less, Kd = 19.5 µM and nH = 1.21 for I344C, and Kd = 3.91 µM and nH = 1.65 for Q98C/I344C. Login to comment 214 ABCC7 p.Phe337Cys ABCC7 p.Leu102Cys In open CFTR channels, internally applied MTS reagents can penetrate far enough into the pore as to modify L102C in TM1 and F337C in TM6. Login to comment 217 ABCC7 p.Ile336Cys ABCC7 p.Arg104Cys Interestingly, amino acid side chains only one to two residues closer to the outer ends of these TMs, R104C in TM1 and I336C in TM6, can be modified by external, but not internal, MTS reagents (Fig. 9). Login to comment 227 ABCC7 p.Thr338Cys ABCC7 p.Ser341Cys ABCC7 p.Phe337Cys Second, whereas cysteines substituted for TM6 residues in the putative narrow pore region-F337C, T338C, and S341C-could be modified by both intracellular and extracellular MTS reagents (El Hiani and Linsdell, 2010), no residues that could be modified from both sides of the membrane were identified in TM1. Login to comment 228 ABCC7 p.Lys95Cys ABCC7 p.Arg104Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys Thus, the side chains of TM1 mutants K95C, Q98C, P99C, and L102C that we identified as accessible to MTS reagents applied from the inside (Fig. 2) were not accessible to MTSET applied to the outside (Fig. 4), whereas R104C, previously shown to be modified by external MTS reagents (Zhou et al., 2008), was not modified by internal MTSES or MTSET (Fig. 2). Login to comment 238 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys Although we have not investigated the state dependence of MTSES modification in TM1 in such great detail, our present results suggest a similar arrangement in which K95C can readily be modified before channel activation (Fig. 5), whereas Q98C, P99C, and L102C are modified rapidly after channel activation (Fig. 3) but very slowly if at all before activation (Fig. 5). Login to comment 242 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Changes in channel function after the addition of the oxidizing agent CuPhe, that were not reversed by removal of this agent, were taken as evidence for the formation of a disulfide bridge between K95C in TM1 and I344C in TM6, and between Q98C in TM1 and I344C in TM6 (Fig. 6). Login to comment 244 ABCC7 p.Met348Cys ABCC7 p.Gln98Cys In contrast, we found no evidence for disulfide bond formation between a pair of introduced cysteine side chains that would be predicted (based on Fig. 9) to be further apart, Q98C in TM1 and M348C in TM6 (Fig. 6, C and D). Login to comment 251 ABCC7 p.Gln98Cys Currently, it is not clear why there are no sites in TM1 that can be accessed from both sides of the membrane in our hands (although it should be noted that Q98C was described as being sensitive to external MTSES in a previous study; Akabas et al., 1994). Login to comment 256 ABCC7 p.Thr338Cys ABCC7 p.Met348Cys ABCC7 p.Lys95Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys For comparison, the MTSES modification rate constant for P99C and L102C (Fig. 3) was similar to that of T338C and S341C in TM6 (El Hiani and Linsdell, 2010) (all between 100 and 150 M1 s1 ), and the modification rate constant for K95C was comparable to, or slightly greater than, that of I344C, V345C, and M348C (El Hiani and Linsdell, 2010) (all between 2,000 and 4,000 M1 s1 ). Login to comment 258 ABCC7 p.Gln98Cys The MTSES modification rate constant for Q98C (440 M1 s1 ; Fig. 3) was somewhat intermediate between these two groups. Login to comment 261 ABCC7 p.Lys95Cys If this is the case, the similar discrepancy between MTSES and MTSET modification rate constants at all sites tested implies that the location of anion-cation discrimination that underlies this discrepancy may lie between the cytoplasmic mouth of the pore and the most accessible cysteine residue, namely, that at K95C. Login to comment 264 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys 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). Login to comment 265 ABCC7 p.Ile340Cys ABCC7 p.Ile340Cys ABCC7 p.Ser877Cys Cysteine side chains can also be cross-linked between residues at the cytoplasmic ends of TM6 and TM12 (Chen et al., 2004), as well as between I340C (TM6) and S877C (TM7) (Wang et al., 2007); however, these findings are more difficult to interpret in structural terms because longer cross-linking molecules were used, and also because the side chain of I340C is not accessible to the aqueous lumen of the pore (Bai et al., 2010; El Hiani and Linsdell, 2010). Login to comment