ABCMdb

PMID: 16766608

Serrano JR, Liu X, Borg ER, Alexander CS, Shaw CF 3rd, Dawson DC
CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore.
Biophys J. 2006 Sep 1;91(5):1737-48. Epub 2006 Jun 9., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Thr338Cys CFTR: Ligand Exchange between a Permeant Anion ([Au(CN)2]2 ) and an Engineered Cysteine (T338C) Blocks the Pore Jose &#b4; R. Serrano,* Xuehong Liu,* Erik R. Borg,* Christopher S. Alexander,* C. Frank Shaw 3rd,y and David C. Dawson* *Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239; and y Department of Chemistry, Illinois State University, Normal, Illinois 61790-4160 ABSTRACT Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET1 and MTSES . Login to comment 2 ABCC7 p.Thr338Cys Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2] produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). Login to comment 19 ABCC7 p.Thr338Cys To test this hypothesis we used a CFTR construct with a cysteine substituted at position 338, T338C CFTR. Login to comment 20 ABCC7 p.Thr338Cys We recently reported that the functional effects of both covalent and noncovalent modification of T338C CFTR were consistent with the hypothesis that a cysteine substituted at this location lies within the anion conduction path (8). Login to comment 23 ABCC7 p.Cys1344Ser ABCC7 p.Cys524Ser ABCC7 p.Cys276Ser ABCC7 p.Cys491Ser ABCC7 p.Cys225Ser ABCC7 p.Cys1458Ser ABCC7 p.Cys1410Ser ABCC7 p.Cys866Ser ABCC7 p.Cys832Ser ABCC7 p.Cys343Ser ABCC7 p.Cys76Ser ABCC7 p.Cys1400Ser ABCC7 p.Cys1395Ser ABCC7 p.Cys1355Ser ABCC7 p.Cys590Leu ABCC7 p.Cys592Leu MATERIALS AND METHODS Mutagenesis and in vitro transcription The Cys-less CFTR construct (C76S, C126S, C225S, C276S, C343S, C491S, C524S, C590L, C592L, C657S, C832S, C866S, C1344S, C1355S, C1395S, C1400S, C1410S, C1458S) was a gift from Drs. Martin Mense and Submitted December 28, 2005, and accepted for publication May 19, 2006. Login to comment 36 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Most importantly, the construct usedin the experiments reported here, T338C on a Cys-less background (T338C/Cys-less), behaved in a manner that was virtually identical to that of T338C/wt; including similar pH titration of macroscopic conductance (8). Login to comment 45 ABCC7 p.Thr338Cys This variable reactivity is preserved in the T338C/Cys-less construct and cannot, therefore, be attributed to an intrapeptide disulfide bond. Login to comment 64 ABCC7 p.Thr338Cys RESULTS T338C CFTR displays enhanced affinity for [Au(CN)2]2 We reported previously that wt CFTR is reversibly blocked by [Au(CN)2] (7), a result that was confirmed and extended to several CFTR mutants by Linsdell et al. (15-17). Login to comment 69 ABCC7 p.Thr338Ala Similar responses to [Au(CN)2] were observed using oocytes expressing T338A CFTR (Fig. 1 C), and the apparent dissociation constants (in mM) were similar for the three constructs: Kwt &#bc; 0.754, KCys-less &#bc; 0.813, KT338A &#bc; 0.754. Login to comment 71 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys In the experiment depicted in Fig. 2 A, after exposure to DTT, exposure of T338C/wt to 100 mM [Au(CN)2] , a concentration of the pseudohalide that produced only modest inhibition of Cys-less CFTR conductance, virtually abolished the T338C/wt CFTR conductance. Login to comment 74 ABCC7 p.Thr338Cys As indicated in Fig. 2 B, profound inhibition of conductance due to T338C/Cys-less CFTR that was not reversed by washing could be achieved with as little as 100 nM [Au(CN)2] , albeit at a slower rate. Login to comment 85 ABCC7 p.Thr338Ala (C) A representative experiment showing the effect of [Au(CN)2] on T338A CFTR conductance. Login to comment 86 ABCC7 p.Thr338Ala After activation by a stimulatory cocktail (Isop1IBMX, hatched bar), an oocyte expressing T338A CFTR channels was exposed to 1 mM DTT (open circles) and then to 10 mM, 100 mM, 1 mM, and 10 mM of [Au(CN)2] (solid circles). Login to comment 96 ABCC7 p.Thr338Cys Fig. 2 C contains an example of an oocyte expressing T338C/Cys-less CFTR in which it was possible to observe high affinity, irreversible block, and lyotropic block by [Au(CN)2] . Login to comment 98 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Over the course of these experiments we found that in oocytes pretreated with a reducing agent (2-ME or DTT) the extent of inhibition of T338C/(wt or Cys-less) FIGURE 2 [Au(CN)2] produced a profound, irreversible reduction of gCl in oocytes expressing T338C CFTR. Login to comment 120 ABCC7 p.Thr338Cys This result suggests that the variable conductance that remains after irreversible block of T338C/Cys-less by micromolar concentrations of [Au(CN)2] represents CFTR channels that are not susceptible to high affinity block but remain susceptible to lyotropic block. Login to comment 121 ABCC7 p.Thr338Ala Block of the residual conductance by [Au(CN)2] was consistent with an apparent dissociation constant of ;0.8 mM, comparable to that seen with wt, T338A, and Cys-less CFTR. Login to comment 122 ABCC7 p.Thr338Cys This result is consistent with the hypothesis that those spontaneously reacting T338C CFTR channels in which the thiol cannot be recovered by exposure to 2-ME or DTT do not differ from wt CFTR as regards their susceptibility to lyotropic block. Login to comment 125 ABCC7 p.Thr338Cys The results described above are consistent with the pattern of reactivity previously reported for T338C CFTR (13). Login to comment 126 ABCC7 p.Thr338Cys T338C channels that have been previously exposed to 2-ME or DTT comprise at least two subpopulations. Login to comment 128 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Exposure of these channels to nanomolar concentrations of [Au(CN)2] produces a profound, irreversible block that accounts for the majority of the inhibition of macroscopic conductance due to T338C/wt or T338C/Cys-less CFTR. Login to comment 136 ABCC7 p.Thr338Cys An oocyte expressing T338C/wt CFTR was exposed to 1 mM 2-ME (open circles); 100 mM [Au(CN)2] (solid circles) irreversibly reduced gCl by ;80%. Login to comment 139 ABCC7 p.Thr338Cys Exposure of an oocyte expressing T338C/wt CFTR to 100 mM [Au(CN)2] resulted in nearly 100% inhibition of the conductance. Login to comment 142 ABCC7 p.Thr338Cys High affinity block of T338C CFTR by [Au(CN)2] was similarly abolished by exposing oocytes to either MTSET1 or MPA, a polar malemide (not shown). Login to comment 143 ABCC7 p.Thr338Ala Exposure of an oocyte expressing a construct bearing a non-Cys substitution at position 338 (T338A CFTR) to IAM (2 mM) was without effect on reversible, lyotropic block by [Au(CN)2] (13). Login to comment 144 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys High affinity block of T338C CFTR conductance: evidence for a ligand-exchange reaction The irreversible block of T338C CFTR conductance by [Au(CN)2] suggests that the anion participates in a high affinity interaction with the substituted cysteine. Login to comment 147 ABCC7 p.Thr338Cys If the reaction between [Au(CN)2] and T338C CFTR occurs via the mechanism depicted above then it should be possible to reverse the reaction by supplying excess cyanide ligand. Login to comment 149 ABCC7 p.Thr338Cys After activating T338C/Cys-less CFTR and treating the oocyte with 2-ME, exposure to 0.5 mM KCN resulted in an 18% reduction of the conductance that was readily reversed by washing with a KCN-free solution (Fig. 5 A). Login to comment 165 ABCC7 p.Thr338Cys Both anions produced only rapidly reversible, lyotropic block of T338C/Cys-less CFTR that was not affected by blocking the thiolate with IAM (not shown). Login to comment 167 ABCC7 p.Thr338Cys 6 and 7 compare the effects of [Au(CN)2] on Cys-less and T338C/Cys-less CFTR channels recorded from detached, inside out patches. Login to comment 176 ABCC7 p.Thr338Cys This observation differs from that reported by Linsdell and Gong (17) for wt CFTR, suggesting that the interaction of [Au(CN)2] with endogenous cysteines not present in the Cys-less construct may account for the effect on open probability. Exposure to [Au(CN)2] produced a strikingly different response in T338C/Cys-less CFTR channels (n &#bc; 10). Login to comment 183 ABCC7 p.Thr338Cys (A) Oocytes expressing T338C/Cys-less CFTR were activated and exposed first to 1 mM 2-ME (open circles) and then to 500 mM KCN (shaded triangles). Login to comment 187 ABCC7 p.Thr338Cys After activation, oocytes expressing T338C/Cys-less CFTR were exposed to 1 mM 2-ME (open circles) and then to 1 mM [Au(CN)2] (solid circles). Login to comment 190 ABCC7 p.Thr338Cys that the more rare, 0.4 pA events could represent a substate of the T338C/Cys-less channel (22). Login to comment 195 ABCC7 p.Thr338Cys The behavior of this patch was consistent with the notion that it contained T338C/Cys-less CFTR channels that were blocked by [Au(CN)2] and a second channel that was unreactive, yet dependent upon PKA and ATP. Login to comment 198 ABCC7 p.Thr338Cys DISCUSSION High affinity block of T338C CFTR by a permeant anion [Au(CN)2] is a very stable coordination compound with a log stability constant (log K) of 37-39 (20,21); where K is given by K &#bc; &#bd;Au&#f0;CN&#de;2 &#bd;Au 1 &#bd;CN 2: (2) Yet, in a solution containing excess [CN] , individual [CN] undergoes exchange at the Au(I) center at the fast exchange limit detected by 13 C-NMR (23). Login to comment 208 ABCC7 p.Thr338Cys Mechanism of irreversible inhibition of CFTR conductance The profound, rapid decrease in the conductance of oocytes expressing T338C CFTR could reflect a decrease in single-channel conductance, a reduction of channel open probability, or some combination of these two effects. Login to comment 212 ABCC7 p.Thr338Cys Taken together, these observations suggest that at least a portion of the decrease in single-channel conductance produced by exposure of T338C CFTR to [Au(CN)2] reflects the deposition of a negative charge within the pore via the mixed ligand complex, Protein-S-[Au-CN] . Login to comment 213 ABCC7 p.Thr338Cys In FIGURE 7 [Au(CN)2] irreversibly blocked T338C/Cys-less CFTR single-channel currents. Login to comment 229 ABCC7 p.Thr338Cys It is important to note, however, that the single-channel studies reported here were conducted using Cys-less CFTR and T338C/Cys-less CFTR to avoid confounding effects resulting from endogenous cysteines that are accessible to reagents applied to the cytoplasmic face of detached patches (10). Login to comment 231 ABCC7 p.Thr338Cys In any single oocyte expressing T338C CFTR that has been previously exposed to 2-ME or DTT, the CFTR channels appear tocompriseamixedpopulation.In somechannelsthecysteineat 338 is in the simple thiolate form, whereas in others the thiol has undergone a chemical reaction that renders the thiolate unavailable for ligand exchange or thiol-disulfide exchange reactions. Login to comment