ABCMdb

PMID: 18167343

Fatehi M, Linsdell P
State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2008 Mar 7;283(10):6102-9. Epub 2007 Dec 31., 2008-03-07 [PubMed]

Sentences

No. Mutations Sentence Comment

59 ABCC7 p.Arg334Cys As shown previously (9, 15), the R334C mutation itself induced inward rectification because of removal of the native positive charge and reduced electrostatic attraction of extracellular Cl- ions. Login to comment 64 ABCC7 p.Arg334Cys In contrast to these results obtained with inclusion of MTS reagents in the pipette solution, we found that MTSES was apparently unable to modify R334C using a pretreatment protocol. Login to comment 66 ABCC7 p.Arg334Cys This lack of effect could reflect that MTSES does not covalently label R334C and so must be present during the experiment to exert its effect, that covalent modification does occur but is relatively transient, or that covalent modification takes place during pipette application but not during pretreatment. Login to comment 69 ABCC7 p.Arg334Cys Following cAMP stimulation, MTSES was able to modify R334C channels and induce increased inward rectification of the I-V relationship (Fig. 1, G and H), indicating that stable covalent modification of this introduced cysteine could take place but was somehow dependent on the activation state of the channels. Login to comment 71 ABCC7 p.Arg334Cys Conformational Change in the Pore on Activation of CFTR MARCH 7, 2008•VOLUME 283•NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6103 Next we asked if this apparent state-dependent access of MTSES was limited to R334C or if a similar effect could be seen with cysteines introduced at other sites in the pore. Login to comment 73 ABCC7 p.Ser341Cys An example is S341C; as shown in Fig. 2A, inclusion of MTS reagents in the pipette solution also gave charge-dependent changes in I-V shape in this mutant, indicating that deposition of charge at this position also alters anion movement in the pore. Login to comment 74 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Ser341Cys In fact, similar charge-dependent effects were observed in R334C, K335C, T338C, and S341C (Fig. 3). Login to comment 75 ABCC7 p.Phe337Cys Another mutant, F337C, became significantly more inwardly rectified in the presence of MTSES but was apparently not affected by inclusion of MTSET in the pipette solution (Fig. 3). Login to comment 77 ABCC7 p.Arg334Cys The state dependence of modification of cysteines introduced at these sites was studied using the MTS pretreatment protocol described above for R334C. Login to comment 78 ABCC7 p.Ser341Cys Again, results from the example mutant S341C are shown in Fig. 2. Login to comment 79 ABCC7 p.Arg334Cys As with R334C (Fig. 1), MTSES induced inward rectification of the I-V relationship (indicating covalent modification of the substituted cysteine) when included in the pipette solution but not when preincubated with the cells. Login to comment 82 ABCC7 p.Arg334Cys Modification of R334C-CFTR by external MTS reagents. Login to comment 86 ABCC7 p.Arg334Cys C, the same experimental protocol in R334C-CFTR illustrates modification by MTSET and MTSES, seen clearly in the IREL-V relationships as changes in current rectification. Login to comment 90 ABCC7 p.Phe337Cys ABCC7 p.Phe337Cys Conformational Change in the Pore on Activation of CFTR 6104 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 10•MARCH , shape when included in the pipette solution or when preincubated with cells (or, in the case of F337C, the same lack of effect) (Fig. 4A). Login to comment 95 ABCC7 p.Arg334Cys Two positively charged maleimide derivatives, MBTA and TMAEM, had similar effects on R334C as those observed with MTSET (Fig. 5), leading to a more linear I-V relationship. Login to comment 100 ABCC7 p.Arg334Cys The effects of MTSES on R334C were mimicked by another negatively charged reagent, pCMBS (Fig. 6). Login to comment 105 ABCC7 p.Ser341Cys Modification of S341C-CFTR by external MTS reagents. Login to comment 114 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Ser341Cys ABCC7 p.Phe337Cys F, wild type (both panels); E, R334C (left); Ⅺ, K335C (left); ‚, F337C (right); ƒ, T338C (right); छ, S341C (right) (mean of data from 3-9 patches). Login to comment 115 ABCC7 p.Phe337Cys All MTS treatments led to significant changes in rectification ratio (p Ͻ 0.05) except MTSET-wild type, MTSES-wild type, and MTSET-F337C. Login to comment 117 ABCC7 p.Thr338Cys We therefore used Au(CN)2 - , a highly permeant anion that has been shown to covalently modify the introduced cysteine in T338C-CFTR to block the permeation pathway (13). Login to comment 119 ABCC7 p.Thr338Cys We therefore reasoned that if Au(CN)2 - entered the pore to modify the introduced cysteine of T338C covalently, only very small currents could be activated by PKA, ATP, and PPi but that currents would subsequently be activated on exposure to CN- . Login to comment 123 ABCC7 p.Thr338Cys In T338C, however, pretreatment with the same concentration of Au(CN)2 - for only 1 min led to the appearance of a KCN-sensitive component of current (Fig. 7A). Login to comment 128 ABCC7 p.Thr338Cys Thus, although it appears that Au(CN)2 - can modify T338C-CFTR with or without cAMP stimulation, the dramatic increase in the proportion of channels apparently modified by Au(CN)2 - when cAMP stimulation is applied concurrently with Au(CN)2 - exposure suggests that the rate of modification is far greater in activated channels than in nonactivated channels. Login to comment 136 ABCC7 p.Arg334Cys Positively charged maleimide derivatives modify R334C-CFTR prior to channel activation. Login to comment 140 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Ser341Cys ABCC7 p.Ser341Cys Conformational Change in the Pore on Activation of CFTR 6106 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 10•MARCH 7, each of R334C, K335C, and S341C, like T338C, the apparent degree of Au(CN)2 - modification as determined by the KCN- sensitive component of the current was significantly enhanced by cAMP stimulation (Fig. 7E). Login to comment 142 ABCC7 p.Phe337Cys In contrast, F337C was only very weakly inhibited by Au(CN)2 - either with or without cAMP prestimulation. Login to comment 143 ABCC7 p.Phe337Cys Alteration of I-V shape in F337C by MTSES (Figs. Login to comment 147 ABCC7 p.Phe337Cys Whatever the explanation, this lack of effect appears to be specific for F337C because the four other reactive cysteine mutants studied could be modified functionally by all reagents tested. Login to comment 154 ABCC7 p.Ser341Cys In the case of MTSET at least, this large organic molecule is able to enter far enough into the pore of nonactivated channels to react with a cysteine substituted for Ser-341, purportedly located in the pore inner vestibule (22). Login to comment 155 ABCC7 p.Arg334Cys In contrast, MTSES cannot even modify R334C in the outer mouth of the pore under these conditions. Login to comment 157 ABCC7 p.Arg334Cys Reactivity of external MTSET and MTSES with R334C-CFTR has been studied previously, and it was shown that following channel activation both of these reagents modified this introduced cysteine only when the channel was closed (18). Login to comment 162 ABCC7 p.Arg334Cys State-dependent modification of R334C-CFTR by external pCMBS. Login to comment 171 ABCC7 p.Arg334Cys This gating cycle also appears to involve changes in accessibility to both MTSET and MTSES, at least as reported by a cysteine substituted for Arg-334 (18), although in this case there is no apparent charge dependence of accessibility. Login to comment 179 ABCC7 p.Thr338Cys Example I-V relationships recorded following pretreatment with 10 ␮M KAu(CN)2 for 5 min (wild type) or 1 min (T338C) is shown. Login to comment 183 ABCC7 p.Thr338Cys The asterisk indicates a significant differencefromcontrolconditionsforthesamechannelvariant(pϽ0.00001).C,KCN-inducedchangesinCFTR macroscopic conductance for individual patches expressing T338C-CFTR, following pretreatment with KAu(CN)2 alone (E) or KAu(CN)2 plus forskolin and IBMX (F) are shown. Login to comment 188 ABCC7 p.Arg334Cys D, an example I-V relationship for R334C pretreated with 10 ␮M KAu(CN)2 plus forskolin (10 ␮M) and IBMX (100 ␮M) for 1 min recorded before (control) and after treatment with 50 ␮M KCN is shown. Login to comment 189 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Ser341Cys ABCC7 p.Phe337Cys E, the mean change in CFTR macroscopic conductance for R334C, K335C, F337C, and S341C following addition of KCN without (white bars) or with (black bars) cAMP pretreatment is shown (mean of data from 4-5 patches). Login to comment 192 ABCC7 p.Arg334Cys Because modification of R334C, which is thought to be located at the outer mouth of the pore, is state-dependent, we suggest that the conformational change associated with channel activation must affect the very outermost part of the pore to influence access of external ions to Arg-334. Login to comment