ABCMdb

PMID: 25190805

Wang W, Roessler BC, Kirk KL
An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening.
J Biol Chem. 2014 Oct 31;289(44):30364-78. doi: 10.1074/jbc.M114.595710. Epub 2014 Sep 4., [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCC7 p.Lys978Cys ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Login to comment 12 ABCC7 p.Lys978Cys Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge-reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. Login to comment 96 ABCC7 p.Glu267Lys ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu The charge-reversal mutations (E267R and K1060E) at these positions clearly had the most dramatic effects on CFTR activity (the E267K mutation also markedly inhibited CFTR activity; see Fig. 5). Login to comment 102 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu CFTR Gating Mechanism 30366 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, Fig. provides indirect but persuasive evidence that the E267R and K1060E charge-reversal mutations reduced channel activity primarily by reducing the rate of channel opening rather than by shortening the open channel bursts or decreasing the single channel conductance. Login to comment 103 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu Shown in Fig. 3, A and B are unitary current recordings of the E267R and K1060E mutants at FIGURE 1. Login to comment 124 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu These micropatch results indicate that the E267R and K1060E charge-reversal mutants have exceptionally low Pos under control and PKI-treated conditions (10-50-fold lower than WT-CFTR) primarily because they have very low channel opening rates, not abnormally brief open channel bursts. Login to comment 127 ABCC7 p.Glu267Cys ABCC7 p.Lys1060Cys As we showed in Fig. 2 the single E267C and K1060C mutations reduced CFTR channel activity especially after PKI addition but not to the extent of the charge-reversal substitutions. Login to comment 128 ABCC7 p.Glu267Cys Subsequent exposure of E267C-CFTR to MTSET rapidly and markedly inhibited the post-PKI current which was reversed by bath addition of DTT (Fig. 4A). Login to comment 129 ABCC7 p.Lys1060Cys Conversely, the same positively charged reagent modestly increased the current mediated by K1060C-CFTR; an effect that also was reversed by DTT (Fig. 4B; mean data in Fig. 4C). Login to comment 131 ABCC7 p.Glu267Cys To test this idea we exposed E267C-CFTR channels to the poorly hydrolyzable AMP-PNP in the continued presence of ATP prior to MTSET addition (Fig. 4D). Login to comment 133 ABCC7 p.Glu267Cys E267C-CFTR channels also were strongly activated by AMP-PNP but remained sensitive to subsequent treatment with MTSET followed by DTT (Fig. 4D). Login to comment 136 ABCC7 p.Glu267Cys These findings support the idea that the accessibility of E267C to thiol modification is reduced in the open channel conformation(s) as would be expected if this position is buried in a helix bundle upon channel opening. Login to comment 138 ABCC7 p.Glu267Lys ABCC7 p.Lys1060Glu Fig. 5, A--C confirm this prediction for an E267K/K1060E double mutant. Login to comment 141 ABCC7 p.Lys978Cys Combining E267 and K1060 Charge-reversal Mutations with a Gain of Function (GOF) Mutation Reveals the Importance of the Tetrahelix Bundle in Mediating Channel Opening in the Absence of ATP Binding or NBD Dimerization-Previously we reported a class of GOF mutation in ICL3 near the base of TM9 that increases both ATP-dependent and ATP-independent channel activity (e.g. K978C (12)). Login to comment 143 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu Here we combined one of these GOF mutations with the E267R and K1060E mutations to test: (i) if a GOF mutation could rescue the ATP-dependent channel activities of the E267 and K1060 charge-reversal mutants and (ii) if bundle formation and the predicted electrostatic interaction also facilitate channel opening in the absence of ATP binding or NBD dimerization. Login to comment 144 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg Regarding the first issue, Fig. 6A shows that introducing the K978C substitution into the E267R mutant substantially restored ATP-dependent channel activity (compare with Fig. 2B). Login to comment 145 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg The E267R/K978C double mutant exhibited normalized control currents and potentiator responses in macropatch experiments that were similar to wild type levels (see legend for mean data). Login to comment 146 ABCC7 p.Lys978Cys This rescue of the ATP-dependent activity of the E267 charge-reversal mutant is perhaps not surprising given that the K978C substitution reduces the free energy difference FIGURE 2. Login to comment 154 ABCC7 p.Glu267Arg B, macroscopic current record for patch containing many E267R-CFTR channels showing very small control current and very large activation by potentiators. Login to comment 156 ABCC7 p.Glu267Arg C, macroscopic record showing that E267R-CFTR channels also are strongly stimulated by the FDA-approved potentiator, VX-770 (10 òe;M). Login to comment 157 ABCC7 p.Lys1060Glu D, K1060E-CFTR channels also exhibit reduced control currents and strong activation by potentiators. Login to comment 163 ABCC7 p.Glu267Gly For all mutants except E267G the relative currents after PKI addition were significantly less than for WT ( p b0d; 0.05) by unpaired t test. Login to comment 171 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu Regarding the second issue, Fig. 6, B-E show that the E267R and K1060E mutations reduced the ATP-free activity of the K978C GOF mutant. Login to comment 174 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg The latter interpretation is further supported by the results in Fig. 7, which show that the E267R mutation also reduced the ATP-free channel activity of K978C/èc;1198-CFTR, a truncation mutant lacking NBD2 but possessing the indicated GOF mutation. Login to comment 176 ABCC7 p.Glu267Arg Introducing the E267R mutation into this construct substantially reduced the basal ATP-independent currents and correspondingly increased the relative stimulation by potentiators (Fig. 7, B, D, and E). Login to comment 177 ABCC7 p.Glu267Lys ABCC7 p.Lys1060Glu Conversely introducing a charge swap double mutation (E267K/K1060E) had no apparent effect on the basal currents and potentiator responses of this truncation construct (Fig. 7, C-E). Login to comment 179 ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg ABCC7 p.Glu267Arg Combining a Catalytic Mutation with the E267R Mutation Rescues ATP-dependent Activity but Also Provides a Clue that R Domain Conformational Changes May Be Coupled to Bundle Formation-We next combined one of the charge-reversal mutations (E267R) with an NBD2 mutation (E1371S) that inhibits ATP hydrolysis at site 2 (Fig. 8). Login to comment 180 ABCC7 p.Glu1371Ser The E1371S mutation stabilizes the NBD dimer in the presence of ATP and greatly prolongs the open channel bursts (39, 40). Login to comment 181 ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg Our interest was to determine if the E267R mutation inhibited the activity of the E1371S catalytic mutant and/or destabilized the NBD dimer in the absence of ATP hydrolysis. Login to comment 182 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg Instead, however, the E267R/ E1371S-CFTR double mutant behaved similarly to the E1371S-CFTR single mutant with respect to: (i) high control currents in the presence of normally saturating ATP and PKA; (ii) negligible responses to potentiators presumably because the currents were already maximal under control conditions; and (iii) very slow deactivation upon ATP removal (঄deactivaton b0e; 5 min), indicative of a very tight NBD dimer (Fig. 8A; compare with FIGURE 3. Login to comment 183 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu Unitary current recordings indicate that the E267R and K1060E mutations do not obviously affect single channel conductance or open channel burst duration. Login to comment 184 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu A and B, micropatch recordings of E267R-CFTR and K1060E-CFTR channels obtained using smaller tip pipettes in which individual channel openings and closings and unitary currents were detectable before potentiator addition. Login to comment 187 ABCC7 p.Lys1060Glu The magnitudes of the unitary currents at this holding potential were not different from those for WT-CFTR as follows: WT, 0.427 afe; 0.003 pA (mean afe; S.E.) measured for 206 openingsin2patches;E267R,0.419afe;0.005pAfor119openingsin3patches; K1060E, 0.431 afe; 0.004 pA for 166 openings in 3 patches. Login to comment 189 ABCC7 p.Lys1060Glu The mean open channel burst durations calculated by cycle time analysis (see"ExperimentalProcedures")were0.7afe;0.1and0.6afe;0.1sforE267R-CFTR (n afd; 6 patches) and for K1060E-CFTR (n afd; 4), respectively. Login to comment 194 ABCC7 p.Glu1371Ser CFTR Gating Mechanism 30370 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, E1371S-CFTR data in Refs. 39, 40). Login to comment 195 ABCC7 p.Glu267Arg We interpret these results to indicate that the E267R mutation has negligible effects on channel activity once a tight NBD dimer has been formed by ATP binding sans hydrolysis. Login to comment 196 ABCC7 p.Glu1371Ser One interesting difference between the single and double E1371S mutants emerged when we assayed these constructs in the absence of bath PKA (Fig. 8, B-D). Login to comment 198 ABCC7 p.Glu1371Ser Indeed, we observed macroscopic currents for E1371S-CFTR channels in the absence of bath PKA that averaged more than 10% of the maximal currents measured after potentiator addition (Fig. 8, C and D). Login to comment 200 ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg Combining the E267R charge-reversal mutation with the E1371S mutation virtually abolished these PKA-independent currents (Fig. 8, B and D). Login to comment 202 ABCC7 p.Glu267Arg ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu ABCC7 p.Lys1060Glu E267R and K1060E Charge-reversal Mutations Strongly Reduce the PKA Sensitivity of Channel Gating-Fig. 9 shows that the E267R and K1060E mutations markedly impacted the PKA sensitivity of channel activation. Login to comment 205 ABCC7 p.Glu267Lys ABCC7 p.Lys1060Glu Importantly, normal PKA sensitivity was restored by introducing charge swap mutations across the putative interface (E267K/K1060E; Fig. 9, D-F). Login to comment 212 ABCC7 p.Glu267Cys ABCC7 p.Lys1060Cys A positively charged thiol modifier strongly inhibits E267C-CFTR currents in an activity-dependent manner but modestly stimulates K1060C-CFTR currents. Login to comment 213 ABCC7 p.Glu267Cys A, E267C-CFTR macropatch record showing strong and rapid inhibition of this cysteine mutant by bath addition of 30 òe;M MTSET and reversal of this inhibition by 5 mM DTT. Login to comment 215 ABCC7 p.Lys1060Cys B, corresponding K1060C-CFTR macropatch record showing modest stimulation of this cysteine mutant by MTSET that also was reversed by subsequent DTT addition. Login to comment 220 ABCC7 p.Glu267Cys D, E267C-CFTR macropatch record showing that MTSET inhibition was very slow when added after the current was stimulated with 2 mM AMP-PNP. Login to comment 235 ABCC7 p.Glu267Cys In addition, the rate of inhibition of the E267C mutant by this reagent was slowed markedly by AMP-PNP, a poorly hydrolysable ATP analog that prolongs open channel bursts and stabilizes the NBD dimer. Login to comment 238 ABCC7 p.Lys1060Thr Seibert et al. (43) provided the first evidence that the K1060 position is relevant to CFTR gating in their single channel analysis of a mutation (K1060T) that reportedly associates with mild disease (i.e. CBAVD). Login to comment 241 ABCC7 p.Glu267Lys ABCC7 p.Lys1060Glu A, macroscopic current record for a double mutant with charge-reversal substitutions at both positions (E267K/K1060E-CFTR). Login to comment 247 ABCC7 p.Glu267Lys ABCC7 p.Glu267Arg Note that the E267K single mutation strongly inhibited channel activity as did the E267R mutation shown in Fig. 2. Login to comment 248 ABCC7 p.Lys1060Glu See also Fig. 2E for K1060E single mutant data. Login to comment 252 ABCC7 p.Lys1060Glu Error bars (S.E.) for WT and E267/K1060E are too small to be seen. Login to comment 254 ABCC7 p.Lys1060Thr This moderate degree of inhibition by the uncharged K1060T substitution is consistent with our findings. Login to comment 256 ABCC7 p.Glu267Ala ABCC7 p.Glu267Arg These authors observed greater inhibition by a charge-reversal mutation than by a neutral substitution at the E267 position (E267R versus E267A), as we showed here in our more detailed mechanistic study. Login to comment 260 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu Interestingly, Billet et al. (27) did report that a double mutant in which the charges were swapped between these sites (E267R/K1060E) behaved like wild type CFTR in their assay, which is consistent with an electrostatic interaction between these residues that impacts CFTR activity. Login to comment 262 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg Disrupting the E267-K1060 Interaction Primarily Impacts Channel Opening, not Burst Duration or NBD Dimer Stability-The charge-reversal mutations at these two positions dramatically decreased the macroscopic currents and apparent single FIGURE 6. Interplay between the charge-reversal mutations at the bundle interface and a previously reported GOF mutation (K978C); evidence that the E267RandK1060EmutationsalsoinhibitATP-freechannelactivity.A,E267R/K978CdoublemutantbehavesmorelikewildtypechannelsinthepresenceofATP inmacropatchexperiments.ConditionswereidenticaltoFig.2A.Themeanpercentcontrolcurrentsofthisdoublemutantwhennormalizedtothemaximalcurrents measuredafterpotentiatoradditionwere94afe;5.4%and82.8afe;3.9%beforeandafterPKIaddition,respectively(nafd;5).Thesevaluesarenotsmaller(infact,somewhat larger) than those for WT-CFTR (see Fig. Login to comment 264 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu B-D, macroscopic records for the indicated K978C single and double mutants showing that the E267R and K1060E substitutions decreased the fractional currents remaining after ATP removal. Login to comment 266 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg E267R/K978C-CFTR was activated strongly by the subsequent addition of 2 mM AMP-PNP in the absence of ATP, as reported previously for the K978C GOF mutant (panels B, C and Ref. 47). Login to comment 273 ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg The lack of effect on the stability of the activated state is consistent with the virtually complete rescue of the ATP-dependent activity of the E267R bundle mutant that we observed when we combined this mutation with the E1371S catalytic mutation. Login to comment 275 ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg The E267R/E1371S double mutant exhibited high steady-state control currents following activation by ATP and PKA, small relative activation by potentiators and very slow deactivation upon ATP washout similar to that reported for the E1371S single mutant. Login to comment 280 ABCC7 p.Lys978Cys A, control macroscopic record showing the substantial control current and correspondingly modest stimulation by potentiator addition for the previously described K978C/èc;1198 truncation mutant lacking NBD2. Login to comment 282 ABCC7 p.Lys978Cys As reported earlier (12), the channel activity of this construct is ATP-independent and is attributable to the presence of the K978C GOF mutation. Login to comment 283 ABCC7 p.Glu267Arg B, macroscopic record showing relatively smaller control current and stronger activation by potentiator addition for a corresponding truncation construct harboring the E267R mutation. Login to comment 289 ABCC7 p.Lys978Cys **, p b0d; 0.01 compared with K978C/èc;1198 by unpaired t test. N is indicated in parentheses. CFTR Gating Mechanism 30374 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, tion between inactive and active conformations in response to ATP binding and NBD dimerization. Login to comment 290 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu The E267-K1060 Interaction Also Promotes CFTR Channel Opening in the Absence of ATP Binding or NBD Dimerization- Combining a previously reported GOF mutation (K978C) with the E267R or K1060E bundle mutations revealed that the latter mutations also inhibit ATP-free channel activity both in the presence and absence of NBD2 (i.e. for the èc;1198-CFTR truncation construct). Login to comment 291 ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg The K978C substitution did restore nearly normal ATP-dependent activity of the E267R mutant as evidenced by high control currents in the presence of ATP and PKA and small relative activation by potentiators. Login to comment 293 ABCC7 p.Lys978Cys Apparently the favorable effect of the K978C mutation on the energetics of channel opening is sufficient to overcome the negative impact of the bundle mutation on gating when ATP binding and NBD dimerization can occur. Login to comment 294 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu Conversely, each of the E267R and K1060E mutations substantially reduced the fractional ATP-independent currents exhibited by the full-length K978C-CFTR construct and also reduced the control currents and increased the relative stimulation by potentiators for the NBD2-deletion construct bearing the GOF mutation (K978C/ èc;1198-CFTR). Login to comment 298 ABCC7 p.Lys978Cys According to this view, GOF mutations such as K978C enhance the channel activity of the èc;1198-CFTR construct lacking NBD2 by allosterically promoting the E267-K1060 interaction at the bundle interface. Login to comment 301 ABCC7 p.Lys978Cys The latter mechanism is supported by our earlier observation that PKA phosphorylation of the R domain markedly increases the channel activity of the NBD2 deletion construct, K978C/èc;1198-CFTR (12). Login to comment 302 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu The E267R and K1060E mutations in ICL2 and ICL4 both strongly reduced the PKA sensitivity of channel activation in two ways. Login to comment 303 ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg First, in PKA titration experiments the single charge-reversal mutants required much more FIGURE 8. Interplay between the E267R mutation and a site 2 catalytic mutation, E1371S. Login to comment 304 ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg A, macroscopic record showing that the E267R/E1371S double mutant exhibited large control currents, very small stimulation by potentiators and very slow deactivation upon ATP removal. Login to comment 306 ABCC7 p.Glu1371Ser These characteristics are similar to those previously reported for the single E1371S catalytic mutant (39, 40). Login to comment 307 ABCC7 p.Glu1371Ser ABCC7 p.Glu267Arg B and C, macroscopic records showing that the E267R substitution reduced the baseline current prior to addition of PKA (units/ml) that can be detected for the E1371S-CFTR construct. Login to comment 313 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu The former interpretation seems likely given that wild type CFTR channels are maximally active at a PKA concentration (200 units/ml) that only partially activated the single E267R and K1060E mutants. Login to comment 321 ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu A-C, macroscopic current records showing that the E267R and K1060E mutants required more PKA than WT-CFTR to achieve only partial activation. Login to comment 325 ABCC7 p.Glu267Lys ABCC7 p.Glu267Arg ABCC7 p.Lys1060Glu ABCC7 p.Lys1060Glu Symbols are means afe; S.E. Ns are 5, 5, 4, and 5 for WT (black symbols), E267R (red), K1060E (blue), and E267K/K1060E (green), respectively. Login to comment 326 ABCC7 p.Lys978Cys CFTR Gating Mechanism 30376 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, observed that the K978C GOF mutation in ICL3 near the base of TM9 increased the PKA sensitivity of channel activation even though this residue is nowhere near a PKA phosphorylation site in the polypeptide sequence (12). Login to comment