ABCMdb

PMID: 22234285

Wang W, Linsdell P
Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating.
Biochim Biophys Acta. 2012 Mar;1818(3):851-60. Epub 2012 Jan 2., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)2 - ions was reduced when ATP concentration was reduced from 1 mM to 10 μM, and modification by MTSES was accelerated when 2 mM pyrophosphate was applied to prevent channel closure. Login to comment 4 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. Login to comment 5 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Gln We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). Login to comment 6 ABCC7 p.Lys464Ala ABCC7 p.Lys95Cys ABCC7 p.Glu1371Gln ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys The rate of modification of Q98C and I344C by both MTSES and Au(CN)2 - was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. Login to comment 44 ABCC7 p.Glu1371Gln ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys Both wild type CFTR and a variant in which all 18 endogenous cysteine residues have been substituted by other inside-out inside-out + ATP and PKA + ATP and PKA B) Q98C + MTSES (6 s) + MTSES (6 s) + MTSES (30 s) + MTSES (30 s) + MTSES (120 s) + MTSES (120 s) C) Q98C/E1371Q 200 pA 500 ms 250 pA 500 ms A) Voltage Protocol -50 -25 0 25 50 V (mV) 500 ms Fig. 1. Login to comment 47 ABCC7 p.Gln98Cys (B) Raw currents carried by Q98C following patch excision from the cell (inside-out), following channel activation with ATP (1 mM) and PKA, and at different times after application of MTSES (200 μM) to the cytoplasmic face of the membrane. Login to comment 48 ABCC7 p.Glu1371Gln ABCC7 p.Gln98Cys (C) Raw currents carried by Q98C/E1371Q under these same conditions. Login to comment 49 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys Note that background (leak) currents are small in Q98C prior to addition of ATP and PKA, whereas in Q98C/E1371Q, as with all constructs bearing the E1371Q mutation, large constitutive currents are observed and are not further increased in amplitude by PKA and ATP (see also Refs. Login to comment 52 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Example timecourses of macroscopic currents (measured at -50 mV during brief voltage excursions from a holding potential of 0 mV) carried by K95C, Q98C, I344C and V345C as indicated, in inside-out membrane patches. Current amplitudes were measured every 6 s following attainment of stable current amplitude after channel activation. Channels were activated with PKA (20 nM) and either a high concentration of ATP (1 mM; in (A) and (C)-(E)) or a low concentration of ATP (10 μM; (B)). Login to comment 54 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Gln In (D) channels also had the NBD1 mutation K464A, and in (E) channels also had the NBD2 mutation E1371Q. Login to comment 55 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys In each panel, MTSES (20 μM for K95C, I344C and V345C, and 200 μM for Q98C; see Materials and methods) 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 58 ABCC7 p.Val510Ala The cys-less version used in the present study also includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane [19]. Login to comment 60 ABCC7 p.Lys464Ala ABCC7 p.Lys95Cys ABCC7 p.Glu1371Gln ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Additional mutations were introduced into the cys-less background using the QuikChange site-directed mutagenesis -200 -150 -100 -50 0 I (pA) Time (s) K95C A) 1 mM ATP -180 -120 -60 0 Q98C -400 -300 -200 -100 0 -200 -150 -100 -50 0 I344C -300 -200 -100 0 -500 -400 -300 -200 -100 0 V345C -250 -200 -150 -100 -50 0 -300 -200 -100 0 -600 -400 -200 0 -750 -500 -250 0 -600 -400 -200 0 -800 -600 -400 -200 0 I (pA) Time (s) 20 µM MTSES 20 µM MTSES 20 µM MTSES200 µM MTSES C) 1 mM ATP + 2 mM PPi E) E1371Q (1 mM ATP) I (pA) Time (s) D) K464A (1 mM ATP) B) 10 µM ATP -100 -75 -50 -25 0 -200 -150 -100 -50 0 -80 -60 -40 -20 0 -80 -60 -40 -20 0 -120 -90 -60 -30 0 -80 -60 -40 -20 0 -60 -40 -20 0 -300 -200 -100 0 I (pA) Time (s) I (pA) Time (s) 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 system (Agilent Technologies, Santa Clara, CA, USA) and verified by DNA sequencing. Login to comment 63 ABCC7 p.Glu1371Gln In some experiments, CFTR channels bearing the NBD2 mutation E1371Q were used. This mutation results in constitutive, high levels of activity when expressed in BHK cells [23,24] (see Fig. 1C). Login to comment 73 ABCC7 p.Gln98Cys In most cases, 20 μM MTSES and 200 nM Au(CN)2 - were used, however, in channels bearing the Q98C mutation these concentrations were increased to 200 μM MTSES and 200 nM Au(CN)2 - because of the lower rate of modification of a cysteine at this position ([15], and Figs. 3, 6 and 7). Login to comment 75 ABCC7 p.Gln98Cys The similar low apparent modification rate for Q98C by Au(CN)2 - (Figs. 6 and 7) suggests that accessibility of this substance is limited by similar factors as that of MTSES. Login to comment 90 ABCC7 p.Gln98Cys Examples of macroscopic currents recorded from Q98C-CFTR under these conditions are shown in Fig. 1B, and examples of timecourses of MTSES-induced current amplitude change are shown in Fig. 2. Login to comment 91 ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Val345Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys As described previously, at K95C and Q98C (TM1) and at I344C and V345C (TM6), current amplitude is decreased by treatment 100 1000 10000 1 mM ATP 10 µM ATP 1 mM ATP + 2 mM PPi K95C Q98C I344C V345C * * ModificationRateConstant(M-1 s-1 )ModificationRateConstant(M-1 s-1 ) A B K95C Q98C I344C V345C 100 1000 10000 Cys-less +K464A +E1371Q * * * * * * Fig. 3. Login to comment 95 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Gln In (B), NBD function has been altered by the mutations K464A (NBD1) and E1371Q (NBD2). Login to comment 100 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Inspection of these example timecourses indicates that, while such manipulations have no effect on the rate of modification in K95C or V345C, the rate of modification is altered in both Q98C and I344C (Fig. 2A-C). Login to comment 101 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Quantification of the mean modification rate constant (as described in Materials and methods) demonstrates that decreasing ATP concentration from 1 mM (Fig. 2A) to 10 μM (Fig. 2B) to decrease channel opening rate significantly decreases the rate of modification in Q98C and I344C (~2.0-fold decrease in modification rate constant; Pb0.01), whereas the rate of modification of K95C and V345C was apparently unaffected (P>0.2) (Fig. 3A). Login to comment 102 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Conversely, treatment with PPi (2 mM; Fig. 2C) to inhibit channel closure and increase open probability significantly increases the rate of modification in Q98C and I344C (2.5-2.8-fold increase in modification rate constant; Pb0.01) but has no effect on the rate of modification of K95C and V345C (P>0.4) (Fig. 3A). Login to comment 103 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys These results suggest that pharmacological manipulation of NBD function results in changes in the accessibility of Q98C and I344C-but not K95C or V345C-to cytoplasmic MTSES. Login to comment 105 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Gln Indeed, previous studies of pore accessibility changes have taken advantage of NBD mutations K464A and E1371Q [10,11]. Login to comment 106 ABCC7 p.Lys464Ala The NBD1 mutation K464A decreases channel opening rate and has been described as slightly decreasing overall open probability at moderate ATP concentrations [9,28,29], whereas mutagenesis of the key catalytic glutamate residue E1371 in NBD2 prevents ATP hydrolysis to cause dramatic prolongation of CFTR channel open times, presumably leading to an increase in open probability [1,30,31]. Login to comment 107 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Gln To alter channel gating by non-pharmacological means, we therefore combined the NBD mutations K464A and E1371Q with each of the four cysteine mutants described above. Login to comment 109 ABCC7 p.Glu1371Gln Constructs including the E1371Q mutation gave large, spontaneously active currents in BHK cells (Fig. 1C), as described previously, and likely have an open probability close to one [23,24]. Login to comment 111 ABCC7 p.Lys464Ala ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys The K464A mutation significantly decreased the rate of MTSES modification at Q98C and I344C (2.5-2.9-fold decrease in modification rate constant; Pb0.005) but had no effect on the rate of modification at K95C or V345C (P>0.5) (Fig. 3B). Login to comment 112 ABCC7 p.Lys95Cys ABCC7 p.Glu1371Gln ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Conversely, the E1371Q mutation significantly increased the rate of MTSES modification at Q98C and I344C (3.0-3.1-fold increase in modification rate constant; Pb0.02) but had no effect on the rate of modification at K95C or V345C (P>0.25) (Fig. 3B). Login to comment 113 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys These results therefore suggest that altering NBD function non-pharmacologically by mutagenesis alters accessibility of Q98C and I344C to cytoplasmic MTSES, whereas accessibility of K95C and V345C are unaffected by NBD-driven channel gating. Login to comment 145 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys In fact, we found that currents carried by K95C, Q98C, and I344C were potently inhibited by much lower concentrations of Au(CN)2 - (200 nM-2 μM; Fig. 6). Login to comment 146 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys Both K95C and I344C were rapidly inhibited by 200 nM Au(CN)2 - (Fig. 6), reflecting a high modification rate constant (Fig. 7). Login to comment 147 ABCC7 p.Gln98Cys For Q98C, the modification rate constant was lower, and experiments were carried out using a higher concentration of Au(CN)2 - (2 μM). Login to comment 148 ABCC7 p.Gln98Cys Interestingly, the modification rate constant for MTSES is also lower for Q98C (Fig. 3), probably reflecting its location more deeply into the pore [15]. Login to comment 149 ABCC7 p.Val345Cys In contrast, V345C was not sensitive to inhibition by low concentrations of Au(CN)2 - , although it appeared to show the same sensitivity as cys-less to the voltage-dependent blocking effects of higher concentrations of Au(CN)2 - described above (data not shown). Login to comment 150 ABCC7 p.Lys464Ala ABCC7 p.Lys95Cys ABCC7 p.Glu1371Gln ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys We therefore compared the rate of Au(CN)2 - inhibition in K95C, Q98C and I344C at two different ATP concentrations (10 μM and 1 mM), as well as in channels also bearing the NBD mutations K464A or E1371Q (Fig. 6). Login to comment 151 ABCC7 p.Lys464Ala ABCC7 p.Lys95Cys ABCC7 p.Glu1371Gln ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Quantification of the mean modification rate constant demonstrated that decreasing ATP -300 -200 -100 0 -400 -300 -200 -100 0 -200 -150 -100 -50 0 -60 -40 -20 0 -120 -80 -40 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 -100 -75 -50 -25 0 -90 -60 -30 0 -90 -60 -30 0 -120 -80 -40 0 A) 1 mM ATP I (pA) I (pA) I (pA) Time (s) 200 nM Au(CN)2 C) K464A (1 mM ATP) D) E1371Q (1 mM ATP) 2 µM Au(CN)2 200 nM Au(CN)2 I (pA) Time (s) Time (s) -200 -150 -100 -50 0 -90 -60 -30 0 -120 -80 -40 0 K95C Q98C I344C B) 10 µM ATP Time (s) Fig. 6. Timecourse of modification by Au(CN)2 - . Login to comment 153 ABCC7 p.Lys464Ala ABCC7 p.Lys95Cys ABCC7 p.Glu1371Gln ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Reporter cysteines (K95C, Q98C, and I344C as indicated) were examined in isolation (A, B) or combined with the NBD mutations K464A (C) or E1371Q (D). Login to comment 154 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys In each panel, Au(CN)2 - (200 nM for K95C and I344C, and 2 μM for Q98C; see Materials and methods) 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 156 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys concentration to 10 μM significantly decreased the rate of Au(CN)2 - modification of Q98C and I344C (1.7-1.8-fold decrease in modification rate constant; Pb0.005) but had no effect on the rate of modification at K95C (P>0.4) (Fig. 7). Login to comment 157 ABCC7 p.Lys464Ala ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys In addition, the K464A mutation significantly decreased the rate of Au(CN)2 - modification of Q98C and I344C (1.4-1.5-fold decrease in modification rate constant with 1 mM ATP; Pb0.005) but had no effect on the rate of modification at K95C (P>0.5) (Fig. 7). Login to comment 158 ABCC7 p.Lys95Cys ABCC7 p.Glu1371Gln ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Conversely, the E1371Q mutation significantly increased the rate of Au(CN)2 - modification at Q98C and I344C (2.8-3.5-fold increase in modification rate constant; Pb0.01) but had no effect on the rate of modification at K95C (P>0.2) (Fig. 7). Login to comment 165 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys For two introduced cysteine residues-K95C in TM1 and V345C in TM6-the rate of modification by cytoplasmic reagents was independent of ATP-dependent channel gating (Figs. 3, 6), suggesting that access to these residues is similar both in open channels and in closed channels. Login to comment 166 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys In contrast, nearby residues (Q98C in TM1, I344C in TM6) showed strongly state-dependent accessibility, both to the large MTSES (Fig. 3) and the small, permeant Au(CN)2 - ion (Fig. 7). Login to comment 167 ABCC7 p.Lys464Ala ABCC7 p.Glu1371Gln The rate of modification at these two sites was significantly decreased by lowering ATP concentration (Figs. 3A, 7) and by the K464A mutation (Figs. 3B, 7), and significantly increased by PPi (Fig. 3A) and the E1371Q mutation (Figs. 3B, 7). Login to comment 168 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Given the well known effects of these manipulations on channel gating and overall open probability, it seems reasonable to suggest that the rate of modification at Q98C and I344C is positively associated with open probability, suggesting that open channels are modified more easily than are closed channels. Login to comment 172 ABCC7 p.Glu1371Gln The very high apparent open probability of E1371Q mutant channels in BHK cells [23,24] means that constructs bearing this mutation likely give a good estimate of the rate of modification of open channels. Login to comment 173 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys As pointed out above, the lack of apparent state-dependence of modification in K95C and V345C suggests that the rate of modification at these sites is similar in closed channels. Login to comment 174 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys However, our measurements of time constants of changes in macroscopic current amplitude in channels that are opening and closing do not allow us to estimate the rate of modification at Q98C and I344C in closed channels. Login to comment 175 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Previously we suggested that the rate of modification of Q98C [15] and I344C [14] is negligible in inactive channels prior to PKA-dependent phosphorylation. Login to comment 176 ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys It is possible that phosphorylation leads to partial opening of the putative gate and a partial increase in access, and ATP-dependent gating then results in a further increase in access through this region. This could allow slow modification of Q98C and I344C in phosphorylated, but closed, channels. Login to comment 180 ABCC7 p.Lys464Ala For example, reducing ATP concentration to 10 μM is expected to decrease open probability around tenfold [25], whereas the K464A mutation is usually reported as having only a minor effect on open probability [9,28,29]. Login to comment 181 ABCC7 p.Lys464Ala ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys Perhaps surprisingly, then, we find the effects of low ATP and the K464A mutation on modification rate constants for Q98C and I344C to be quantitatively similar and in the range of 1.5 to 3-fold (Figs. 3, 7). Login to comment 201 ABCC7 p.Lys95Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys While K95C, Q98C and I344C were rapidly inhibited by low concentrations of cytoplasmic Au(CN)2 - (Fig. 6), V345C showed similar Au(CN)2 - sensitivity as cys-less CFTR (data not shown). Login to comment 202 ABCC7 p.Val345Cys While the reasons why V345C is apparently not modified by Au(CN)2 - are not clear, we have previously found that not all pore-lining cysteine side chains that can be modified by MTS reagents can also be modified by Au(CN)2 - [14]. Login to comment 203 ABCC7 p.Val345Cys Furthermore, it has been shown that V345C is insensitive to external Au(CN)2 - , although cysteines substituted for other nearby side chains are inhibited under similar conditions [33]. Login to comment