ABCMdb

PMID: 19114635

Wang X, Bompadre SG, Li M, Hwang TC
Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel.
J Gen Physiol. 2009 Jan;133(1):69-77., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCC7 p.Gly551Asp Recently we found that the disease-associated mutation G551D (the single-amino acid substitution of glycine to aspartate in the signature sequence of NBD1) abolishes the ATP-dependent gating of the channel (Bompadre et al., 2007). Login to comment 14 ABCC7 p.Gly551Asp The importance of the signature sequence is attested by how drastically the G551D mutation affects the gating of the channel and by the severity of the disease phenotype associated with this mutation, but little is known about its specific role in CFTR gating. Login to comment 17 ABCC7 p.Gly551Asp The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. Login to comment 18 ABCC7 p.Gly551Asp We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Login to comment 19 ABCC7 p.Gly551Asp Here, we report that micromolar [Cd2+ ] can dramatically increase the activity of G551D-CFTR in the absence of ATP. Login to comment 20 ABCC7 p.Gly551Ala This effect of Cd2+ is not seen in wild-type channels or in G551A. Login to comment 21 ABCC7 p.Gly551Asp Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd2+ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd2+ . Login to comment 22 ABCC7 p.Ser549Cys ABCC7 p.Gly551Cys ABCC7 p.Leu548Cys The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR`s NBD1, show robust response to Cd2+ . Login to comment 23 ABCC7 p.Gln552Cys ABCC7 p.Thr547Cys ABCC7 p.Arg553Cys On the other hand, negligible effects of Cd2+ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd2+ binding to the gate. Login to comment 48 ABCC7 p.Gly551Asp For the G551D mutant, because it does not respond to ATP, we used the current in the absence of ATP (which is the same as in the presence of 1 mM ATP) as control. Login to comment 49 ABCC7 p.Ser549Cys ABCC7 p.Gly551Cys For the G551C and S549C mutants, because they are ATP dependent, we used the current in the presence of 1 mM ATP as control. Login to comment 50 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ser549Cys The fold increase of the current in the presence of Cd2+ was normalized to the maximal fold increase for each mutant (100 μM for G551D, 10 μM G551D, and 5 μM for S549C). Login to comment 53 ABCC7 p.Gly551Asp Here, we found that "soft" metal ions such as Cd2+ and Zn2+ can dramatically increase the activity of G551D-CFTR with a micromolar-apparent affinity. Login to comment 54 ABCC7 p.Gly1349Asp This effect of Cd2+ was not seen with the wild-type (WT) channels or the corresponding mutation, G1349D, at the signature sequence of NBD2. Login to comment 55 ABCC7 p.Gly551Cys The apparent affinity for Cd2+ was further increased when glycine 551 was converted to cysteine, but the effect of Cd2+ was mostly abolished when the G551 residue was substituted by an alanine. Login to comment 58 ABCC7 p.Gly551Asp Consistent with this idea, pretreatment of the G551D-CFTR with the sul- Figure 1. Login to comment 67 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Because 100 μM Cd2+ potentiates G551D activity by ~20-fold, we estimate that the Po of G551D-CFTR is 0.08 ± 0.03 under this condition. Login to comment 68 ABCC7 p.Gly551Asp From patches with fewer than five simultaneous channel opening steps, we calculated the open time of G551D channels in the presence of 100 μM Cd2+ to be 2.4 ± 0.4 s (n = 10). Login to comment 69 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Then the opening rate of G551D-CFTR in the presence of 100 μM Cd2+ is ~0.036 ± 0.015 s-1 , a near fourfold increase (3.6 ± 1.8) of the opening rate of G551D-CFTR in the absence of Cd2+ . Login to comment 70 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Although Cd2+ increases the activity of G551D-CFTR, Cd2+ shows little effect on G1349D-CFTR (n = 5) (not depicted), a mutation of the corresponding glycine residue in the signature sequence of NBD2, indicating that this effect of Cd2+ is specific for the glycine-to-aspartate mutation at NBD1. Login to comment 74 ABCC7 p.Gly551Asp Thus, like ATP-dependent gating for WT-CFTR, activation of G551D mutant channels by Cd2+ requires pre-phosphorylation of the channel by PKA, and the Cd2+ -induced currents can be readily and reversibly inhibited by inh-172. Login to comment 75 ABCC7 p.Gly551Asp Because the activation of G551D-CFTR channels by Cd2+ is more effective than Zn2+ , we focused our studies on this particular cation. Login to comment 79 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp R E S U LT S Cd2+ Increases the Activity of G551D-CFTR We studied the effect of different divalent cations, including Cd2+ , Ca2+ , Ni2+ , and Zn2+ , on G551D-CFTR channels in excised inside-out membrane patches from transiently transfected Chinese hamster ovary cells. Login to comment 80 ABCC7 p.Gly551Asp Fig. 2 A shows a real-time recording of G551D-CFTR channels in an excised patch that have been activated with 1 mM ATP and PKA (not depicted) before being exposed to different metal ions. Login to comment 84 ABCC7 p.Gly551Asp Because CFTR channels can only be opened by ATP after they have been phosphorylated by PKA, we tested whether the effect of Cd2+ on G551D-CFTR is also phosphorylation dependent. Login to comment 86 ABCC7 p.Gly551Asp As shown in Fig. 3 A, Cd2+ only increased the current of G551D-CFTR after the channels had been pre-phosphorylated by PKA (n = 5). Login to comment 87 ABCC7 p.Gly551Asp Previously, we estimated the Po of G551D channels to be 100-fold smaller than the maximal Po of WT channels, ~0.004 ± 0.001 with a mean open time of 367 ± 42 ms Figure 2. Login to comment 88 ABCC7 p.Gly551Asp Effect of different metal ions on G551D-CFTR and WT-CFTR currents. Login to comment 89 ABCC7 p.Gly551Asp (A) 10 μM Cd2+ and 10 μM Zn2+ potentiate G551D-CFTR ATP-independent currents, but 1 mM Ca2+ and 10 μM Ni2+ have little effect on the currents. Login to comment 93 ABCC7 p.Gly551Asp For G551D-CFTR, 100 μM Cd2+ increases the current by 21.38 ± 4.19-fold (n = 6), but the current response is not quite saturated. Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Cys ABCC7 p.Gly551Cys On the other hand, 10 μM Cd2+ already generates a maximal response for G551C-CFTR, with a maximal fold increase of 7.4 ± 0.3 (n = 5) compared with the currents generated by 1 mM ATP. Fitting the dose-response relationships with the Hill equation yields a K1/2 of 14.6 ± 6.3 μM and 3.29 ± 0.66 μM for G551D and G551C, respectively. Login to comment 96 ABCC7 p.Gly551Ala ABCC7 p.Gly551Cys Like the G551C mutant, G551A-CFTR remains responsive to ATP. Login to comment 97 ABCC7 p.Gly551Ala ABCC7 p.Gly551Cys However, the effect of Cd2+ on G551A-CFTR is negligibly small compared with that of G551C-CFTR (Fig. 5 A vs. Fig. 4 B). Login to comment 98 ABCC7 p.Gly551Ala ABCC7 p.Gly551Cys This difference between G551A and G551C was quantified in Fig. 5 B, where we compared the current generated by 1 mM ATP with the current generated by 10 μM Cd2+ for these two mutants. Login to comment 102 ABCC7 p.Ser549Cys A representative S549C-CFTR current recording is shown in Fig. 6 A. Cd2+ elicited macroscopic current even at sub-micromolar [Cd2+ ]. Login to comment 104 ABCC7 p.Thr547Cys In contrast, 10 μM Cd2+ shows negligible effect on the T547C mutant (Fig. 6 B). Login to comment 106 ABCC7 p.Gln552Cys ABCC7 p.Thr547Cys ABCC7 p.Arg553Cys It appears that when cysteine is engineered outside the signature sequence (i.e., T547C and R553C) or at the C-terminal end of the signature sequence (i.e., Q552C), ATP remains a much better ligand than Cd2+ (e.g., Fig. 6 B). Login to comment 107 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ser549Cys ABCC7 p.Gly551Cys ABCC7 p.Gly551Cys ABCC7 p.Leu548Cys However, for L548C, S549C, and G551C, the specificity of the ligand is altered so that Cd2+ becomes more effective at gating Cd2+ Is More Potent on G551C than on G551D We considered two possible mechanisms for the effect of Cd2+ on G551D-CFTR. Login to comment 109 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Alternatively, the G551D mutation may induce protein conformational changes, and these structural changes enable Cd2+ to enhance the activity of G551D-CFTR. Login to comment 111 ABCC7 p.Gly551Cys To differentiate these two possibilities, we first mutated the glycine at position 551 to cysteine. Login to comment 113 ABCC7 p.Gly551Asp ABCC7 p.Gly551Cys Fig. 4 shows representative traces of G551D (A) and G551C (B) in the presence of different [Cd2+ ]. Login to comment 114 ABCC7 p.Gly551Cys Note that 5 μM Cd2+ induces a higher G551C-CFTR current than 1 mM ATP, despite that this mutation retains responsiveness to ATP. Login to comment 115 ABCC7 p.Gly551Asp Figure 3. Functional characterization of the Cd2+ -dependent effect in G551D-CFTR. Login to comment 116 ABCC7 p.Gly551Asp (A) Activation of G551D-CFTR by Cd2+ is phosphorylation dependent. Login to comment 117 ABCC7 p.Gly551Asp Note that in the same patch, Cd2+ only increases the activity of G551D-CFTR after the channels are activated by PKA and ATP. Login to comment 118 ABCC7 p.Gly551Asp (B) A CFTR-specific blocker, inh-172, can inhibit the G551D-CFTR current induced by 200 μM Cd2+ . Login to comment 122 ABCC7 p.Gly551Asp The G551D-CFTR channels were exposed to 1 mM MTSEA for a short period of time (<1 min), and MTSEA was subsequently washed out. Login to comment 126 ABCC7 p.Gly551Asp To further test this hypothesis, we made a G551D-CFTR construct with 16 out of the 18 endogenous cysteine residues replaced with serines (the 2 cysteine residues, C590 and C592, left unchanged are essential for protein expression). Login to comment 127 ABCC7 p.Gly551Asp Cd2+ can no longer activate this G551D/16-Cys-less channel (not depicted). Login to comment 132 ABCC7 p.Ser549Cys ABCC7 p.Gly551Cys The Binding Partner of Cd2+ Is Likely To Be a Cysteine Residue The micromolar affinity of Cd2+ in activating G551C or S549C mutants raises the possibility that some endogenous cysteine(s) or histidine(s) may participate in form- Figure 4. Login to comment 133 ABCC7 p.Gly551Asp ABCC7 p.Gly551Cys Representative current traces of G551D-CFTR (A) and G551C-CFTR (B) in the presence of different [Cd2+ ]. Login to comment 134 ABCC7 p.Gly551Asp ABCC7 p.Gly551Cys The Cd2+ dose-response relationships for G551D-CFTR (C) and G551C-CFTR (D) were fitted with the Hill equation, y = min + (max-min)/ [1+ (K1/2/[x])n )] (smooth curves). Login to comment 135 ABCC7 p.Gly551Asp ABCC7 p.Gly551Cys The fold increase of the current in the presence of Cd2+ was normalized to the maximal fold increase for each mutant (G551D: 21.38 ± 4.19-fold, 100 μM Cd2+ ; G551C: 7.4 ± 0.3, 10 μM Cd2+ ). Login to comment 136 ABCC7 p.Gly551Asp ABCC7 p.Gly551Cys G551D: K1/2 = 14.6 ± 6.3 μM and n = 1.03 ± 0.34; G551C: K1/2 = 3.29 ± 0.66 μM and n = 1.89 ± 0.52. Login to comment 137 ABCC7 p.Gly551Ala ABCC7 p.Gly551Cys Figure 5. Comparison of Cd2+ and ATP-induced currents between G551A and G551C mutants. Login to comment 138 ABCC7 p.Gly551Ala (A) Representative current trace of G551A-CFTR. Login to comment 139 ABCC7 p.Gly551Ala Although G551A-CFTR remains ATP dependent, Cd2+ fails to increase the activity of the channels. Login to comment 140 ABCC7 p.Gly551Ala ABCC7 p.Gly551Cys (B) The ratio of currents induced by 10 μM Cd2+ and those with 1 mM ATP for G551C-CFTR and G551A-CFTR. Login to comment 143 ABCC7 p.Gly551Asp As described above, the opening rate of G551D-CFTR is increased by Cd2+ . Login to comment 144 ABCC7 p.Gly551Cys For G551C-CFTR, this effect of Cd2+ on the opening rate likely also occurs. Login to comment 145 ABCC7 p.Gly551Cys The open time for G551C-CFTR in the absence of ATP is 202.6 ± 29.6 ms (n = 3). Login to comment 148 ABCC7 p.Gly551Asp ABCC7 p.Ser549Cys ABCC7 p.Gly551Cys ABCC7 p.Leu548Cys D I S C U S S I O N Here, we show that micromolar concentrations of Cd2+ can dramatically increase the activity of G551D-CFTR, a disease-associated mutant, as well as G551C-CFTR, L548C, and S549C-CFTR, in the absence of ATP. Login to comment 157 ABCC7 p.Ser549Cys (A) Representative S549C-CFTR current recording in the presence of different [Cd2+ ]. Login to comment 158 ABCC7 p.Thr547Cys (B) Representative T547C current recording in the presence of 1 mM ATP or 10 μM Cd2+ . Login to comment 159 ABCC7 p.Ser549Cys (C) Dose-response relationship for S549C-CFTR fitted with the Hill equation (solid line); K1/2 = 2.4 ± 0.8 μM and n = 1.12 ± 0.39. Login to comment 165 ABCC7 p.Gly551Asp First, as shown in Fig. 3 A, gating of G551D-CFTR by Cd2+ , like ATP-dependent gating of WT-CFTR, requires prior phosphorylation of the channels by PKA. Login to comment 166 ABCC7 p.Gly551Asp Second, G551D-CFTR currents induced by Cd2+ can be inhibited by inhibitor-172, a gating modifier that has been shown to inhibit ATP-dependent gating of WT-CFTR (Ma et al., 2002; Caci et al., 2008). Login to comment 167 ABCC7 p.Gly551Asp Third, normal gating of WT-CFTR channels involves interactions of ATP with the signature sequence of NBD1, as exemplified by the G551D mutation that completely eliminates the ATP-dependent gating (Bompadre et al. 2007). Login to comment 168 ABCC7 p.Thr547Cys ABCC7 p.Arg553Cys Fig. 6 demonstrates that the Cd2+ -dependent gating also involves the signature sequence because engineering cysteine residues framing the signature sequence (i.e., T547C and R553C) did not confer this effect of Cd2+ . Login to comment 174 ABCC7 p.Gly551Asp That a mutation in the signature sequence of NBD1 (i.e., G551D) renders a channel completely irresponsive to ATP further supports the critical role of ABP2 in catalyzing channel opening (Bompadre et al., 2007). Login to comment 176 ABCC7 p.Gly551Asp MTSEA abolishes the effect of Cd2+ on G551D-CFTR. Login to comment 177 ABCC7 p.Gly551Asp G551D-CFTR channels were activated with 1 mM ATP plus PKA (not depicted). Login to comment 181 ABCC7 p.Gly551Asp Pathophysiological Implications The disease-associated mutant G551D has an ~100-fold smaller Po than WT channels. Login to comment 182 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Many compounds (Amaral and Kunzelmann, 2007) and ATP analogues (Cai et al., 2006; Bompadre et al., 2007) have been found to potentiate the activity of G551D-CFTR channels, but none of them could increase the activity of G551D-CFTR to WT levels. Login to comment 184 ABCC7 p.Gly551Asp Interestingly, the 20-fold increase of the G551D-CFTR current by Cd2+ is by far the most effective potentiation demonstrated for this disease-associated mutant. Login to comment 187 ABCC7 p.Gly551Asp Nevertheless, our findings open the door for rational drug design that can greatly benefit cystic fibrosis patients carrying the G551D mutation. Login to comment 188 ABCC7 p.Gly551Asp These studies suggest that the signature sequence of NBD1 can be a drug target for rescuing the dysfunctional G551D channels. Login to comment 189 ABCC7 p.Gly551Asp It is worth noting that the significance of our results in future drug design should hold even if this Cd2+ -dependent gating for G551D-CFTR and normal ATP-dependent gating of WT channels turn out to use different gating machinery. Login to comment 202 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D and G1349D, two CF-associated mutations in the signature sequence of CFTR, exhibit distinct gating defects. Login to comment 216 ABCC7 p.Ser549Cys ABCC7 p.Gly551Cys First, the apparent affinities for G551C and S549C are at low micromolar range, supporting the idea that multiple cysteines are involved in coordinating Cd2+ . Login to comment 217 ABCC7 p.Gly551Asp Second, a thiol-specific reagent, MTSEA, can abolish the effect of Cd2+ on G551D. Login to comment 218 ABCC7 p.Gly551Asp Third, based on the Monod-Wyman- Changeux model (Scheme 2) for allosteric modulation of protein function by ligand binding (Monod et al., 1965), we can conclude that when Cd2+ binding yields an ~30-fold increase of the gating constant (i.e., KЈ/K = 30, where KЈ = kЈo/kЈc and K = ko/kc), as in the case of G551D, thermodynamics dictates that the binding affinity for the open state has to be 30-fold higher than that for the closed state (i.e., Kd/KdЈ = 30, where Kd = koff/ kon and KdЈ = kЈoff/kЈon). Login to comment 226 ABCC7 p.Ser549Cys Our preliminary data (not depicted) indeed suggest that this may be the case because removing all six cysteines in NBD2 does not seem to affect the action of Cd2+ on S549C-CFTR. Login to comment 271 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. 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