ABCMdb

PMID: 23709221

Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PubMed]

Sentences

No. Mutations Sentence Comment

20 ABCC7 p.Thr338Ala ABCC7 p.Ser1141Ala Infrequent subconductance behavior is seen in some CFTR mutants, such as T338A/Cand S1141A-CFTR (7, 12). Login to comment 21 ABCC7 p.Arg347His ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Ala However, subconductance states are dominant events with short burst durations in CFTR channels bearing known salt bridge mutations, such as R352A, R347H, D993R, and D924R (13, 14). Login to comment 23 ABCC7 p.Asp1152Ala Mutation D1152A in the inner vestibule of TM12 resultsinchannelsthatfrequentlyexhibitthes1ands2openstates as well as the f open state (7). Login to comment 25 ABCC7 p.Arg334Cys For example,R334C-CFTRchannelsroutinelyopentothes1andthen s2statesandtransitiontothefstatejustbeforeclosing(15).Hence, it is reasonable to believe that CFTR channel pore opening might involve a complicated sequence of multiple steps leading to the occupancy of a stable, full open state. Login to comment 49 ABCC7 p.Cys590Leu ABCC7 p.Cys592Leu All cRNAs for single channel recording were prepared from constructs encoding WT-CFTR or Cys-less CFTR (16C 3 S, C590L, C592L) in the pGEMHE vector, which was kindly provided by Dr. D. Gadsby (Rockefeller University) as reported previously (21). Login to comment 72 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys ABCC7 p.Asp993Cys Open burst durations and closed durations were measured from single channel recordings of WT-, R352C-, D993C-, and R352C/D993C-CFTR, and then histograms and fits of them with single exponential functions were generated with IGOR (WaveMetrics, Inc., Lake Oswego, OR) to determine time constants for open burst durations (঄o, also called beta) and closed durations (঄c, also called ॷ) for all of the constructs. Login to comment 82 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg RESULTS Arg347 Forms a Salt Bridge with Asp924 but Does Not Stabilize the Full Open State-Although Cotten and Welsh first reported that arginine 347 of TM6 forms a salt bridge with aspartic acid 924 of TM8, their results suggested that the double mutation R347D/D924R rescued the channel to a stable open state that exhibits a smaller single channel amplitude, which is reminiscent of the s2 open state of WT-CFTR (14). Login to comment 89 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg347Lys ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg ABCC7 p.Arg347Ala A, representative current samples of WT-, R347A-, R347D-, D924R-, R347K-, and R347D/D924R-CFTR were recorded from excised inside-out patch from Xenopus oocytes with 150 mM Clafa; symmetrical solution in the presence of 1 mM Mg-ATP and 50 nM PKA at VM afd; afa;100 mV (n afd; 4-6 for each mutant). Login to comment 94 ABCC7 p.Arg347Asp ABCC7 p.Arg347Ala R347A-CFTR showed a very long and stable s1 state with very brief openings to s2 or f states, whereas R347D-CFTR only exhibits a long stable s1 state and appears to never get out of s1 (at the resolution of our recording apparatus), as if introduction of negative charge at this position confers electrostatic repulsion with other negative charges in the native channel and thereby greatly interferes with the ability to go beyond the s1 state. Login to comment 95 ABCC7 p.Arg347Lys R347K-CFTR, retaining the positive charge of arginine, showed behavior similar to WT-CFTR (afa;0.72 afe; 0.02 pA, n afd; 7) but with a slightly larger single channel amplitude (afa;0.89 afe; 0.01 pA, n afd; 4, p b0d; 0.05). Login to comment 96 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Arg347Ala D924R-CFTR exhibits all three open states in contrast to R347A- and R347D-CFTR, although the stability of the open state is compromised; indeed, the fractional occupancies of both s1 and s2 states are greatly increased in this mutant (Fig. 2B). Login to comment 97 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg The charge-swapping double mutant R347D/D924R-CFTR exhibited a long stable s2 state with occasional brief openings to s1 and f. Login to comment 99 ABCC7 p.Arg347Lys In all of the Arg347 and Asp924 mutants described above, other than R347K, transitions to the f state did not lead to stable occupancy of that state. Login to comment 101 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg347Ala ABCC7 p.Arg347Ala In addition, breakingthissaltbridgedisruptedthestabilityofthes2andfstates but did not significantly affect s1; therefore, both R347A and R347D showed long stable s1 states, although R347A endeavored to reach the s2 and f states but failed to maintain them, whereas R347D completely lost the ability to open to s2 and f state. Login to comment 104 ABCC7 p.Arg352Glu In our prior studies, we found that R352E-CFTR can open to all three conductance levels, with all open states being unstable. Login to comment 105 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu Similar results were found for D993R-CFTR, but nearly wild type-like behavior, including stable openings to the f state, was recovered in the R352E/D993R double mutant (see Fig. 3A). Login to comment 109 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Arg347Lys ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu ABCC7 p.Arg347Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala We therefore hypothesized that Arg347 might also interact with Asp993 to rescue the CFTR channel pore to a stable f state and tested this hypothesis in three double mutants; TABLE 1 Summary of the effects of mutations studied Mutant Main features of open bursts Impact on f state R347A Emphasizes s1 state, brief transitions to s2 and f Can reach f but not stable R347D Emphasizes s1 state, no transitions to s2 and f Cannot reach f D924R Brief transitions to all conductance levels Can reach f but not stable R347K Wild type-like Wild type-like R347D/D924R Emphasizes s2 state, rare and brief transitions to f Can reach f but not stable R352E Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable D993R Opens to all 3 levels, but none are stable Can reach f but not stable R352E/D993R Wild type-like, with increased transitions to s1 and s2; slightly reduced single-channel conductance Wild type-like R352E/D924R Opens to all 3 levels, but none are stable Can reach f but not stable R347D/D993R Very stable s2; rare and brief transitions to both s1 and f Can reach f but not stable R347A/R352A Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable R347D/D924R/D993R Opens to all 3 levels; s1 much more stable than in WT, s2 relatively stabilized, f unstable Can reach f but not stable R347D/D924R/R352E/D993R Primarily flickers between s2 and f; s1 much more stable than in WT, slightly reduced single channel conductance Can reach f but not stable FIGURE 3. Login to comment 111 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu A, representative current samples of R352E/D993R-, R352E/D924R-, and R347D/D993R-CFTR recorded from excised inside-out patches with the same conditions as Fig. 2 (n afd; 3-6 for each mutant). Login to comment 114 ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu As noted above, R352E/D993R exhibited a prominent full open state similar to WT-CFTR (13), suggesting that the R352E/D993R salt bridge can fully rescue the CFTR channel pore to normal behavior (aside from a slight decrease in single channel conductance). Login to comment 115 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg352Glu Whereas the single channel behavior of R352E/D924R was similar to that of R352E alone, with multiple unstable open states, suggesting that Arg352 and Asp924 do not interact, R347D/D993R was much more like R347D/D924R, with the s2 state dominant (compare Figs. 3 and 2). Login to comment 116 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg R347D/ D993R-CFTR is able to transition to the f state but sojourns there are even more brief than those seen for the R347D/ D924R. Login to comment 121 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg In the R347D/ D924R mutant, the positive charge at Arg347 is no longer available to interact with Asp993 . Login to comment 122 ABCC7 p.Arg347Asp ABCC7 p.Asp993Arg Similarly, in the R347D/D993R mutant, the positive charge at Arg347 is no longer available to interact with Asp924 . Login to comment 123 ABCC7 p.Arg347Asp Therefore, we asked whether replacing the negative charge at both Asp924 and Asp993 with a positive charge would allow strong enough interactions with R347D to enable channels to go to the f state. Login to comment 124 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg This was tested in the triple mutant R347D/D924R/D993R (Fig. 4, A and B). Login to comment 125 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg Unlike the two double mutants described above (R347D/D924R and R347D/ D993R), the triple mutant exhibited roughly equal occupancy of s1,s2,andfstates;theoccupancyofthes2statewasnotasstableas in either double mutant. Login to comment 129 ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala As we show in Fig. 4, R347A/ R352A-CFTR behaves just like R352A-CFTR, opening to all three conductance states with little stability of either state, as we reported before. Login to comment 131 ABCC7 p.Arg352Ala In R352A-CFTR, Arg347 can still interact with Asp924 and Asp993 . Login to comment 141 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu However, the quadruple mutant R347D/D924R/D993R/R352E did not completely rescue WT behavior (Fig. 4, A and B). Login to comment 146 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg ABCC7 p.Asp924Arg ABCC7 p.Asp993Arg ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala Representative current samples of R347A/R352A-, R347D/D924R/D993R-, and R347D/D924R/D993R/R352E-CFTR were recorded under the same conditions as in Fig. 3 (n afd; 5-6 for each mutant) (A). Login to comment 150 ABCC7 p.Val510Ala MTS Reagents Exhibit No Effects on Single Channel Amplitude of WT-CFTR and Cys-less V510A-CFTR-Because the Arg352 -Asp993 interaction appeared to make the largest contribution to stabilizing the f state, we asked whether forcing these residues to interact would lead to channels that were latched into the open state. Login to comment 152 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys We generated R352C/D993C-CFTR and exposed the channels to MTS-2-MTS; MTS-2-MTS was chosen for this experiment because it leads to cross-linking of cysteines at a distance of b03;4.6 &#c5;, which is within the average distance for known salt bridges in a varietyofproteins(13).Priortostudieswiththedoublemutant,we also investigated each single mutant and their responses to monofunctional sulfhydryl-modifying reagents. Login to comment 155 ABCC7 p.Val510Ala We further added a secondary mutation to generate Cys-less V510A-CFTR in order to improve expression (9, 25). Login to comment 156 ABCC7 p.Val510Ala Because it was reported that Cys-less CFTR showed channel behavior similar to that of WT-CFTR with a few nominal differences (9, 26-28), we first used mutants generated on the WT-CFTR background and then confirmed the results in mutants generated on the Cys-less V510A-CFTR background. Login to comment 157 ABCC7 p.Val510Ala Representative data indicating that charged monofunctional MTS reagents modified the activity of WT-CFTR or Cys-less V510A-CFTR when applied cytoplasmically in excised, inside-out patches are shown in supplemental Fig. 1. Login to comment 160 ABCC7 p.Val510Ala Supplemental Fig. 1B shows Cys-less V510A-CFTR in the absence and presence of MTS reagents; not surprisingly, MTS reagents exhibited no effects on either single channel amplitude or NPo in the Cys-less channel. Login to comment 161 ABCC7 p.Val510Ala Based on these results, we resolved to use both WT-CFTR and Cys-less V510A-CFTR as backgrounds to test the consequences of modification of engineered cysteines. Login to comment 162 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Glu ABCC7 p.Arg352Ala ABCC7 p.Asp993Cys Recovery of Charge at R352C and D993C Rescued Channel Stability in the Full Open State-R352C-CFTR exhibited single channel behavior similar to that previously reported for R352A-, R352Q-, and R352E-CFTR (13). Login to comment 163 ABCC7 p.Arg352Cys A representative recording is shown in Fig. 5A, taken from one membrane patch bearing R352C-CFTR before and after exposure to MTSEAaf9; and after wash out. Login to comment 165 ABCC7 p.Arg352Cys Prior to exposure to MTSEAaf9; , R352C-CFTR exhibited multiple conductance states, including closed (c) and s1, s2, and f open states. Login to comment 166 ABCC7 p.Arg352Cys R352C-CFTR channels opened to all open states for very short durations (Fig. 5B). Login to comment 167 ABCC7 p.Arg352Cys After exposure to MTSEAaf9; , R352C-CFTR channels exhibited mainly the f state with much longer mean burst duration and appearance of the s1 and s2 states as rare events, indicating recovery of open state stability (Fig. 5B). Login to comment 171 ABCC7 p.Arg352Cys In contrast, deposition of negative charge at R352C-CFTR by exposure to MTSESafa; did not alter channel behavior (supplemental Fig. 2A). Login to comment 172 ABCC7 p.Arg352Ala As a control, we show that R352A-CFTR was not sensitive to modification by MTS reagents (supplemental Fig. 2B). Login to comment 174 ABCC7 p.Arg352Cys We note that in R352C-CFTR on the WT-CFTR background, exposure to MTSEAaf9; and MTSETaf9; led to an increase in NPo (Fig. 5A), reflecting modification of endogenous cysteines. Login to comment 175 ABCC7 p.Val510Ala This added effect was lost on the Cys-less V510A background. Login to comment 176 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys In contrast to these results for R352C-CFTR, the stability of single channel opening in D993C-CFTR was rescued to mimic that of WT-CFTR by exposure to MTSESafa; (but not MTSEAaf9; or MTSETaf9; ), leading to significantly increased mean burst duration (supplemental Fig. 3B). Login to comment 177 ABCC7 p.Asp993Cys Exposure to MTSESafa; also significantly decreased the conductance of the f state of D993C-CFTR (supplemental Fig. 3B). Login to comment 179 ABCC7 p.Arg352Cys ABCC7 p.Val510Ala ABCC7 p.Val510Ala ABCC7 p.Asp993Cys We repeated the above experiments in R352C/Cys-less V510A-CFTR and D993C/ Cys-less V510A-CFTR to further rule out the possibility of any endogenous cysteines being involved in the process. Login to comment 181 ABCC7 p.Val510Ala These effects were removed only upon application of the reducing agent DTT and indicate that the Cys-less V510A-CFTR background was identical to the WT-CFTR background with respect to these experiments. Login to comment 182 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys ABCC7 p.Asp993Cys A Bifunctional MTS Reagent Can Latch R352C/D993C-CFTR into the Full Open State Even after Washout of ATP-We hypothesized that the CFTR channel pore could be latched into the open state by cross-linking the two cysteines at R352C and D993C. Login to comment 183 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys We first tested the effects of monofunctional reagents MTSETaf9; , MTSEAaf9; , and MTSESafa; on the double mutant R352C/D993C-CFTR. Login to comment 184 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys None of these reagents rescued salt Dynamic Modulation of the CFTR Pore by Salt Bridges JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20763 bridge function to stabilize channel behavior in R352C/D993C-CFTR in terms of stable openings to the f state (data not shown). Login to comment 185 ABCC7 p.Arg352Cys ABCC7 p.Val510Ala ABCC7 p.Asp993Cys Before applying MTS-2-MTS to the R352C/D993C-CFTR double mutant, we first tested the effects of this bifunctional linker on WT-CFTR (Fig. 6A) and Cys-less V510A-CFTR (Fig. 6B). Login to comment 186 ABCC7 p.Val510Ala MTS-2-MTS did not change the single channel amplitude of either channel but decreased NPo of WT-CFTR without changing NPo of Cys-less V510A-CFTR. Login to comment 187 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys We then examined the effects of MTS-2-MTS on R352C-D993C-CFTR (on the WT-CFTR background); a representative experiment is shown in Fig. 7. Login to comment 188 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys In the presence of ATP and PKA, prior to the addition of MTS-2-MTS, R352C/D993C-CFTR exhibited low open probability, unstable openings to the f state, and occasional subconductance open states. Login to comment 191 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys It seems likely that this reflects the fact that there are several possible consequences of exposing the double mutant to MTS-2-MTS, including covalent modification of R352C and D993C separately by two MTS-2-MTS molecules within each CFTR protein. Login to comment 195 ABCC7 p.Arg352Cys Deposition of positive charge at R352C improved stability of the open state. Login to comment 196 ABCC7 p.Arg352Cys A, sample traces from R352C-CFTR recorded from one patch under control conditions (top trace, ATP af9; PKA) and in the presence of 100 òe;M MTSEAaf9; (middle trace, ATP af9; PKA af9; MTSEAaf9; ) and then after washout with a large volume of intracellular solution and the subsequent addition of ATP and PKA alone (bottom trace, ATP af9; PKA) in excised inside-out membrane patches. Login to comment 198 ABCC7 p.Arg352Cys All traces were recorded at VM afd; afa;100 mV. B, comparison of mean burst duration of R352C in the absence of MTSEAaf9; (ATP af9; PKA only) (afa;MTSEA) and in the presence of MTSEAaf9; with ATP af9; PKA (af9;MTSEA). Login to comment 201 ABCC7 p.Val510Ala Effects of 100 òe;M MTS-2-MTS on WT-CFTR (A) and Cys-less V510A-CFTR (B) at VM d1d; d1a;100 mV. A, MTS-2-MTS decreased NPo but had no effect on single channel amplitude of WT-CFTR. Login to comment 202 ABCC7 p.Val510Ala B, the cross-linker had no effect on either NPo or single channel amplitude of Cys-less V510A-CFTR. Login to comment 209 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys ABCC7 p.Asp993Cys The free energy change èc;èc;G between WT-CFTR and R352C/D993C-CFTR was afa;1.508 kcal/mol, which suggests that R352C and D993C interact with each other when CFTR is in the open state (supplemental Fig. 5). Login to comment 213 ABCC7 p.Thr338Ala ABCC7 p.Lys335Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala We conclude that the subconductance states in CFTR probably also represent pore conformational change for the following reasons: 1) the CFTR channel pore forms from one polypeptide as a monomer and only bears one permeation pathway (12); 2) the s1 and s2 states occur as rare events in some point mutations, such as T338A/Cand K335A/C-CFTR, which do not appear to affect gross pore architecture, whereas they are frequent events in CFTR channels bearing salt bridge mutations, such as R352A- and R347A-CFTR, as discussed above; 3) mutations at sites involved in salt bridges (such as Arg347 , Arg352 , Asp924 , and Asp993 ) result in much more frequent occupancy of subconductance states; 4) mutations at sites involved in salt bridges (such as Arg347 and Arg352 ) lead to greatly altered sensitivity to pore blockers (7, 13); and 5) the subconductance behavior is not affected by different concentrations of Clafa; or by changes in membrane potential (12, 16). Login to comment 218 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys A, effects of 100 òe;M MTS-2-MTS on R352C-D993C-CFTR. Login to comment 220 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys B, MTS-2-MTS failed to functionally modify R352C/D993C-CFTR when applied when the channel was in the closed state. Login to comment 226 ABCC7 p.Arg347Ala R347A-CFTR single channel traces clearly show that the channel first opens from the c to s1 state and then attempts to further open to the s2 and f state; we never saw the channel directly open from c to s2 or f in these mutants. Login to comment