ABCMdb

PMID: 21059651

Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.His950Arg ABCC7 p.Ser768Arg More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Login to comment 22 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys Our recent study also demonstrated that a K190C/S mutation from CL1 enhances ATP-independent channel opening induced by a K978C/P/S mutation from CL3 (17). Login to comment 37 ABCC7 p.Gly551Asp It has been reported that PKA does regulate an NBD1-NBD2 interaction (12, 13) and that PKA can regulate ATP-independent gating in CFTR constructs with G551D (20) and constructs lacking NBD2 (⌬1198) (17, 21, 22). Login to comment 42 ABCC7 p.Ser660Ala In addition, removal of residues 760-783 or 817-838 (NEG2) or much of the R domain (⌬708-835/S660A) from CFTR eliminates PKA dependence of channel activity (26-28). Login to comment 62 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala ⌬R-S660A-CFTR was provided by Michael Welsh (University of Iowa) (31). Login to comment 69 ABCC7 p.Val510Ala To improve expression, cells expressing Cys-free CFTR-based constructs inserted with V510A (32) were grown for 1-2 days at 24 °C and then for 2-5 h at 37 °C before measurements. Login to comment 91 ABCC7 p.Ser768Cys RESULTS Disulfide Cross-linking of the R Domain to CL3-If Ser768 inhibits channel activity by interacting with outwardly facing residues from CL3, disulfide cross-linking of S768C to a corresponding cysteine inserted in CL3 will be expected to suppress channel activity. Login to comment 92 ABCC7 p.Ser768Cys As a negative control, S768C cannot form an inhibitory disulfide bond with an inwardly facing cysteine mutated in CL3. Login to comment 94 ABCC7 p.Ser768Cys ABCC7 p.Ser737Cys S768C or S737C were fixed as an anchor point to search other inhibitory targets from CL3 (Fig. 2A). Login to comment 95 ABCC7 p.His950Cys ABCC7 p.Ser768Cys S768C/H950C was a representative example (Fig. 2B). Login to comment 99 ABCC7 p.His950Cys ABCC7 p.His950Cys ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys B-D, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing mutants H950C/S768C (B), H950C (C), and S768C (D) by using a ramp protocol (Ϯ80 mV). Login to comment 108 ABCC7 p.His950Cys ABCC7 p.Ser768Cys F, unitary currents from a H950C/S768C construct in the presence of 20 ␮M diamide (b) and 4 mM DTT (c). Login to comment 115 ABCC7 p.His950Cys ABCC7 p.Ser768Cys In contrast, both diamide and DTT had no effect on H950C and S768C CFTR constructs (Fig. 2, C and D). Login to comment 116 ABCC7 p.His950Cys ABCC7 p.Ser768Cys These observations clearly suggest that a disulfide bond may be formed between S768C and H950C. Login to comment 118 ABCC7 p.His954Cys ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys ABCC7 p.Lys951Cys ABCC7 p.Ser955Cys Supporting this argument, internal diamide also inhibited activity of a mutant S768C/K951C, S768C/H954C, or S768C/ S955C, and inhibition was reversed by 4-6 mM DTT (Fig. 2E). Login to comment 119 ABCC7 p.His954Cys ABCC7 p.Lys951Cys ABCC7 p.Ser955Cys In sharp contrast, both diamide and DTT had no effect on such CFTR constructs as K951C, H954C, and S955C. Login to comment 120 ABCC7 p.His950Cys ABCC7 p.Val769Cys Furthermore, channel activity of V769C/H950C was also inhibited by diamide (Fig. 2E), suggesting that phosphorylation of Ser768 may not affect the CL3-R interface. Login to comment 121 ABCC7 p.Ser768Cys ABCC7 p.Val956Cys ABCC7 p.Lys946Cys However, S768C could not form an inhibitory disulfide bond with V956C (inwardly facing) or K946C, possibly as a result of a long distance or a poor relative orientation (Fig. 2E). Login to comment 122 ABCC7 p.His954Cys ABCC7 p.Ser737Cys ABCC7 p.Ser737Cys ABCC7 p.Ser737Cys ABCC7 p.Ser955Cys ABCC7 p.Gln958Cys Similarly, diamide also suppressed channel activity of mutants S737C/H954C, S737C/S955C, and S737C/Q958C, and suppression was reversed by DTT (Fig. 2E). Login to comment 125 ABCC7 p.His950Cys ABCC7 p.His954Cys ABCC7 p.Ser768Cys ABCC7 p.Ser737Cys Fig. 3 demonstrates that a CFTR construct with a single cysteine S768C, S737C, H950C, or H954C exhibited a clear single band no matter whether diamide or DTT was added. Login to comment 126 ABCC7 p.His950Cys ABCC7 p.His950Cys ABCC7 p.His954Cys ABCC7 p.His954Cys ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys ABCC7 p.Ser737Cys ABCC7 p.Ser737Cys In sharp contrast, CFTR constructs with a cysteine pair (Cys-free background), S737C/H950C, S737C/H954C, S768C/H950C, and S768C/H954C, exhibited an additional cross-linked (X-linked) band because it was induced by diamide but was weakened by DTT. Login to comment 127 ABCC7 p.His950Cys ABCC7 p.His954Cys ABCC7 p.Val956Cys ABCC7 p.Val956Cys In contrast, the H950C/V956C mutant exhibited no X-linked band possibly because of a poor relative orientation between H954C and V956C. Login to comment 128 ABCC7 p.His950Cys ABCC7 p.His950Cys ABCC7 p.His954Cys ABCC7 p.His954Cys ABCC7 p.Ser768Cys ABCC7 p.Ser737Cys Therefore, a disulfide bond can be formed between H950C (or H954C) and S768C or between H954C (or H950C) and S737C. Login to comment 129 ABCC7 p.His950Cys ABCC7 p.Ser768Cys In order to address if the disulfide bond changes the gating kinetics, a two-channel recording of the H950C/S768C construct was done. Login to comment 138 ABCC7 p.Ser768Ala Fig. 4C shows that S768A was dramatically activated by curcumin in the presence of ATP, but PKA failed to continue to potentiate channel activity. Login to comment 140 ABCC7 p.His950Ala It is very exciting that H950A was also greatly activated by curcumin after pretreatment of ATP, but subsequent PKA further increased channel activity (Fig. 4D). Login to comment 141 ABCC7 p.Lys946Ala ABCC7 p.Lys951Ala ABCC7 p.Ser955Ala ABCC7 p.Gln958Ala Similarly, curcumin also activated mutants K946A, K951A, S955A, and Q958A to a different extent in the presence of ATP (Fig. 4E). Login to comment 143 ABCC7 p.Ser737Ala ABCC7 p.His954Ala ABCC7 p.His954Cys ABCC7 p.Ser737Cys In contrast, curcumin had no such effect on S737A and H954A mutants, suggesting that they may be weak inhibitory residues, although disulfide cross-linking of S737C to H954C strongly inhibited channel activity (Figs. 2E and 4E). Login to comment 144 ABCC7 p.Ser737Ala ABCC7 p.His954Ala Because curcumin increased initial channel activity of most mutants at the R-CL3 interface after ATP was present, it is reasonable that subsequent PKA dependence was greatly reduced except for S737A and H954A. Login to comment 145 ABCC7 p.Ser768Ala ABCC7 p.Ser955Ala Especially, S768A and S955A, together with CFTR-⌬R, completely removed PKA dependence (Fig. 4F). Login to comment 146 ABCC7 p.Ser768Ala ABCC7 p.His950Ala In order to further investigate if ATP is required for the effects of curcumin on S768A and H950A, curcumin was first applied to their intracellular sides before ATP was introduced. Login to comment 150 ABCC7 p.Ser768Ala ABCC7 p.His950Ala It is interesting that both H950A and S768A still needed more PKA to be fully activated in this case (Fig. 5D). Login to comment 156 ABCC7 p.Ser768Asp Thus, if primary phosphorylation of Ser768 inhibits channel activation by curcumin in the presence of ATP, S768D, which is equivalent to phosphorylated Ser768 , should also dampen channel activation by curcumin. Login to comment 157 ABCC7 p.Ser768Asp However, Fig. 6A demonstrates that S768D was also activated by curcumin with ATP. Login to comment 159 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.Ser768Cys In addition, because S768D is negatively charged, an electrostatic attraction between S768D and Lys946 or Lys951 may be impossible because modification of S768C with MTSCE (negatively charged) or MTSET (positively charged) failed to change channel activity (supplemental Fig. S1, A and B). Login to comment 160 ABCC7 p.Ser768Asp On the other hand, S768D is a strong H-bond acceptor (Table 1). Login to comment 162 ABCC7 p.His950Cys ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys ABCC7 p.Ser955Cys Because disulfide cross-linking of S768C to H950C inhibited more channel activity than that of S768C to S955C (Fig. 2E), it is more possible for His950 to form an inhibitory H-bond with Ser768 . Login to comment 163 ABCC7 p.Ser768Arg This notion was supported by the observation that S768R, a strong H-bond donor, failed to be activated by curcumin even in the presence of ATP (Fig. 6B). Login to comment 165 ABCC7 p.Ser768Cys Supporting this proposal, modification of S768C with MTSEA (a very strong H-bond donor (33)) also inhibited 25% of channel activity after the channel was activated by ATP and PKA followed by PKI to block further phosphorylation (supplemental Fig. S1, C and D). Login to comment 166 ABCC7 p.Ser768Thr ABCC7 p.Ser768Thr Because S768T strongly enhanced PKA dependence in the presence of ATP and curcumin, S768T may be a stronger H-bond donor FIGURE 4. Effects of curcumin on CFTR constructs at the R-CL3 interface in the presence of ATP. Login to comment 167 ABCC7 p.Ser768Ala ABCC7 p.His950Ala A-D, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing WT (A), ⌬R (B), S768A (C), and H950A (D). Login to comment 173 ABCC7 p.His950Arg ABCC7 p.His950Asp In agreement with a putative H-bond between Ser768 and His950 , H950R was also activated by curcumin upon ATP treatment, but H950D was not (Fig. 6, C and D). Login to comment 175 ABCC7 p.His950Gln ABCC7 p.His950Gln Because H950Q reduced sensitivity to PKA even in the presence of ATP and curcumin, H950Q may be a stronger H-bond acceptor (Fig. 6D). Login to comment 176 ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.Ser768Arg ABCC7 p.His950Asp What is more, curcumin also activated H950R/ S768R and H950D/S768D constructs in the presence of ATP (Fig. 6E) because two strong proton donors or acceptors cannot form an H-bond (Table 1). Login to comment 177 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.His950Arg More importantly, even if both H950R and S768D could be activated by curcumin with ATP involvement (Fig. 6, A and C), H950R/S768D was silent in response to curcumin even in the presence of ATP (Fig. 6E), suggesting that a strong electrostatic attraction between H950R and S768D prohibit the channel from activation. Login to comment 178 ABCC7 p.Ser768Arg ABCC7 p.His950Asp Consistent with this notion, H950D/S768R was also not activated by curcumin with ATP (Fig. 6E). Login to comment 181 ABCC7 p.Ser737Asp It is reasonable that Ser737 may not form an inhibitory H-bond because S737D was not activated by curcumin even if ATP was added (Fig. 6F), further suggesting that Ser737 may be a weak inhibitory site. Login to comment 184 ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.Ser768Arg ABCC7 p.Ser768Arg Fig. 7A shows that H950R/S768R was activated by ATP only, even without curcumin, but H950R or S768R was not (Fig. 7C). Login to comment 185 ABCC7 p.Lys978Cys ABCC7 p.His950Arg ABCC7 p.Ser768Arg More importantly, H950R/S768R completely removed PKA dependence of channel activity (Fig. 7, A and D) no matter whether K978C, which promotes the channel opening without ATP, was inserted and accelerated channel activation by ATP (Fig. 7B) or not. Login to comment 186 ABCC7 p.Ser768Asp ABCC7 p.His950Asp In contrast, ATP failed to activate construct H950D/S768D, although an H-bond cannot be formed between two strong proton acceptors (Fig. 7C). Login to comment 187 ABCC7 p.Ser768Asp ABCC7 p.His950Asp This result may be due to an endogenous Fe3ϩ binding between S768D and H950D, which also prevented the channel from opening (30). Login to comment 188 ABCC7 p.Ser768Asp ABCC7 p.His950Asp Supporting this hypothesis, Fig. 7C and supplemental Fig. S2 clearly demonstrate that the mutant S768D/H950D can be much activated by ATP once 5 mM EDTA was added to remove the endogenous Fe3ϩ in the channel. Login to comment 189 ABCC7 p.Ser768Asp ABCC7 p.His950Asp In contrast, H950D and S768D could not be dramatically activated by ATP only even in the presence of 5 mM EDTA (Fig. 7C). Login to comment 190 ABCC7 p.Ser768Asp ABCC7 p.His950Asp Fig. 7D and supplemental Fig. S2 further show that the presence of EDTA clearly weakened the PKA dependence of H950D/S768D channel activity. Login to comment 191 ABCC7 p.His950Arg ABCC7 p.Ser768Arg Thus, a strong electrostatic expulsion between H950R/D and S768R/D promoted channel opening by ATP alone. Login to comment 192 ABCC7 p.Ser737Asp ABCC7 p.Gln958Asp ABCC7 p.Gln958Arg ABCC7 p.Ser737Arg It is reasonable that both Q958R/S737R and Q958D/S737D were not activated by ATP because Ser737 was a weak inhibitory site (Fig. 7C). Login to comment 193 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.His950Ala ABCC7 p.Ser768Arg ABCC7 p.His950Asp Unlike H950R/S768R and H950D/S768D, which exerted an electrostatic interaction between the R domain and CL3, H950A, S768A, S768D, and H950R were not apparently activated by ATP only (Fig. 7C) but more sensitive to PKA than WT CFTR (Fig. 7D). Login to comment 196 ABCC7 p.Ser768Ala ABCC7 p.His950Ala A and B, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing H950A (A) and S768A (B). Login to comment 207 ABCC7 p.Ser768Ala In contrast, an apparent open probability of S768A or H956A was as low as 0.0001 to 0.0005, comparable with that of WT CFTR. Login to comment 210 ABCC7 p.Ser768Asp It is interesting that S768D also promoted channel opening by ATP (Fig. 8, D and E). Login to comment 211 ABCC7 p.Ser768Asp Because S768D is equivalent to phosphorylated Ser768 , this finding suggests that phosphorylated Ser768 should not form an inhibitory H-bond. Login to comment 213 ABCC7 p.Ser768Ala ABCC7 p.His950Arg ABCC7 p.His950Ala ABCC7 p.Ser768Arg Unlike H950A or S768A/D, an apparent open probability of H950R/S768R was higher (Po(app) ϭ 0.0042) than that of WT CFTR even in the absence of ATP, and ATP binding further increased channel opening (Po(app) ϭ 0.198) (Fig. 8, D and E). Login to comment 214 ABCC7 p.Ser768Asp ABCC7 p.His950Asp Similarly, H950D/S768D also exhibited an increased apparent open probability (Po(app) ϭ 0.0132) in the presence of 5 mm EDTA, and ATP continued to promote channel opening (Po(app) ϭ 0.0452) (Fig. 8, D and E). Login to comment 223 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp ABCC7 p.His950Ala However, the activation time became significantly shorter for H950A, S768A, and S768D (Fig. 9, B-E). Login to comment 225 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp It is very interesting that apparent basal activity of S768A was not so high and was comparable with that seen with S768D (Fig. 9, C and D). Login to comment 228 ABCC7 p.Ser768Asp ABCC7 p.His950Arg Fig. 9G shows that an open probability of WT CFTR was very low (0.00004) in the resting cells, no FIGURE 6. Effects of curcumin on PKA-dependent activity of His950 and Ser768 mutants with different H-bond donors and acceptors. A and C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing S768D (A) and H950R (C). Login to comment 234 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.His950Ala ABCC7 p.His954Ala ABCC7 p.Ser768Arg ABCC7 p.Ser768Arg ABCC7 p.Ser768Arg ABCC7 p.Ser768Arg ABCC7 p.Lys946Ala ABCC7 p.Lys951Ala ABCC7 p.Ser955Ala ABCC7 p.Gln958Ala ABCC7 p.Ser768Thr ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.His950Asp ABCC7 p.His950Gln Error bars, S.E. TABLE 1 Potential roles in hydrogen bonding at the CL3-R domain interface Note that mutants whose channel activity was increased by curcumin in the presence of ATP are highlighted in boldface type. Residues Role in H-bond Mutants Arg Strong donor H950R, S768R, H950R/S768R, H950R/S768D, H950D/S768R Asp Strong acceptor H950D, S768D, H950D/S768D, H950R/S768D, H950D/S768R Thr, Gln, Ser, His Donor/Acceptor H950Q, S768T, WT Ala Negative control K946A, H950A, K951A, H954A, S955A, Q958A, S737A, S768A, ⌬R Inhibition of CFTR by Ser768 JANUARY 21, 2011•VOLUME 286•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2177 matter whether cAMP was present or not in the extracellular perfusate. Login to comment 235 ABCC7 p.His950Ala In contrast, H950A exhibited a higher basal open probability (Po(app) ϭ 0.0120), but extracellular cAMP did not exert further influence. Login to comment 236 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp For S768A or S768D, a basal open probability was only a little higher (Po(app) ϭ 0.0004) than that of WT CFTR, and extracellular cAMP failed to increase channel activity significantly. Login to comment 237 ABCC7 p.Ser768Ala Accordingly, disruption of the putative H-bond by the S768A/D mutation could not promote basal channel opening even if there was enough ATP in the resting cell. Login to comment 239 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp Although S768A/D disrupted hydrogen bonding with His950 , the Fe3ϩ binding was still strong, and S768D may enhance the metal binding affinity (30). Login to comment 240 ABCC7 p.His950Ala Supporting this possibility, H950A increased basal channel opening to a higher level, possibly by weakening Fe3ϩ binding. Login to comment 244 ABCC7 p.His950Arg ABCC7 p.His950Cys ABCC7 p.Ser768Arg ABCC7 p.Ser768Cys Although thiol-specific disulfide cross-linking of S768C to H950C or nearby cysteines inserted in CL3 inhibited channel activity primarily by stopping the channel from opening, an electrostatic expulsion between S768R/D and H950R/D clearly promoted channel opening even in the absence of ATP. Login to comment 246 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp Furthermore, both S768A and S768D increased sensitivity of CFTR activity to ATP, curcumin, and PKA phosphorylation. Login to comment 248 ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.Ser768Arg ABCC7 p.His950Asp Finally, both H950R and S768D promoted channel opening by ATP followed by curcumin or PKA phosphorylation, but H950D or S768R could not. Login to comment 253 ABCC7 p.Ser768Asp Finally, even if S768D activity is as low as WT CFTR activity under basal conditions (25), functional studies based on the whole-cell recordings cannot distinguish phosphorylated Ser768 from the unphosphorylated residue if both inhibit channel activity. Login to comment 255 ABCC7 p.Lys978Cys ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.Ser768Arg ABCC7 p.Ser768Arg PKA-dependent activity of His950 and Ser768 mutants with different H-bond donors and acceptors. A and B, activation of H950R/S768R (A) and H950R/S768R/K978C (B) before and after ATP (1. Login to comment 259 ABCC7 p.Ser768Asp ABCC7 p.His950Asp For the H950D/S768D construct in the presence of 5 mM EDTA (n ϭ 6-7); **, p Ͻ 0.05 compared with the absence of EDTA. Login to comment 262 ABCC7 p.Ser768Asp Fig. 6 clearly demonstrates that S768D, which is equivalent to phosphorylated Ser768 , is different from WT CFTR in the inside-out patch. Login to comment 263 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp First, both S768A and S768D mutants could be activated by ATP followed by curcumin, but WT CFTR could not even be activated in the presence of ATP (Figs. 4 and 6). Login to comment 264 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp Second, both S768A and S768D were more sensitive to PKA phosphorylation than WT CFTR (Fig. 7D). Login to comment 265 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp Third, the open probabilities of both S768A and S768D were increased by ATP, whereas that of WT CFTR was not (Fig. 8). Login to comment 267 ABCC7 p.Ser768Asp In fact, not all Ser768 may be phosphorylated in the resting cell because the basal in vivo open probability of S768D was a little higher (Po ϭ 0.0004) than that of WT CFTR (Po ϭ 0.00004) (Fig. 9). Login to comment 268 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* ABCC7 p.Ala462Phe Role of Curcumin in Normal CFTR Gating-A previous study (21) showed that curcumin activates mutant CFTR channels, such as G551D, W1282X, ⌬1198, and A462F. Login to comment 272 ABCC7 p.Ser768Ala ABCC7 p.His950Ala A-C, unitary currents across inside-out membrane patches excised from transfected HEK-293T cells expressing WT CFTR (A), S768A (B), and H950A (C) in the absence and presence of ATP (1. Login to comment 282 ABCC7 p.Ser768Ala ABCC7 p.His950Ala However, Figs. 4 and 5 clearly demonstrate that regulation of normal channel gating by curcumin required ATP because ATP binding to the NBDs promoted channel opening of H950A and S768A mutants (Fig. 8). Login to comment 288 ABCC7 p.His950Cys ABCC7 p.Ser768Cys Second, diamide-induced disulfide bond cross-linking of S768C to H950C or its neighboring cysteines, which was confirmed by the SDS-PAGE mobility (Fig. 3), inhibited channel activity (Fig. 2). Login to comment 291 ABCC7 p.Ser768Asp ABCC7 p.His950Arg ABCC7 p.Ser768Arg ABCC7 p.His950Asp In agreement with a proposal of His950 as an H-bond acceptor and Ser768 as an H-bond donor, S768D and H950R mutants were more sensitive to ATP or curcumin or PKA phosphorylation than S768R and H950D (Figs. 6-8). Login to comment 293 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp ABCC7 p.His950Ala A-D, unitary currents across cell-attached membrane patches of transfected HEK-293T cells expressing WT CFTR (A), H950A (B), S768A (C), and S768D (D). Login to comment 304 ABCC7 p.Ser768Cys ABCC7 p.Ser768Cys Error bars, S.E. Inhibition of CFTR by Ser768 2180 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 3•JANUARY of S768C with MTSEA (a strong H-bond donor (33)), but not with MTSCE (a strong H-bond acceptor), inhibited channel activity, whereas modification with MTSET failed to (supplemental Fig. S1). Login to comment 305 ABCC7 p.His950Arg ABCC7 p.Ser768Arg Finally, both proton donors cannot form an inhibitory H-bond between H950R and S768R. Login to comment 306 ABCC7 p.His950Arg ABCC7 p.Ser768Arg Instead, an electrostatic expulsion between H950R and S768R dramatically increased the ATP-independent open probabilities and sensitivity to ATP and PKA (Figs. 7 and 8). Login to comment 307 ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.His950Asp ABCC7 p.His950Asp In contrast, channel activity was not potentiated by an electrostatic expulsion between H950D and S768D until EDTA was added to remove potential endogenous Fe3ϩ binding to S768D and H950D, although both proton acceptors cannot form an inhibitory H-bond (Fig. 7 and 8). Login to comment 311 ABCC7 p.Ser737Ala In contrast, the S737A mutation failed to promote channel opening by ATP and curcumin, although it was also closed to CL3 (Figs. 2-4). Login to comment 313 ABCC7 p.His954Ala Similarly, His954 could not form an inhibitory H-bond with Ser768 because H954A was not dramatically activated by a combination of ATP and curcumin (Fig. 4E). Login to comment 314 ABCC7 p.Lys946Ala ABCC7 p.Lys951Ala ABCC7 p.Ser955Ala ABCC7 p.Gln958Ala On the other hand, K946A, K951A, S955A, and Q958A still promoted channel opening by ATP followed by curcumin (Fig. 4, E and F). Login to comment 323 ABCC7 p.Ser768Ala ABCC7 p.His950Ala It is exciting that both S768A and H950A increased PKA sensitivity no matter whether curcumin was present or not (Figs. 4-7). Login to comment 324 ABCC7 p.Ser768Asp Because S768D also reduced PKA dependence of channel activity even without curcumin involvement (Figs. 6 and 7), most of the Ser768 in WT CFTR may not be phosphorylated in the excised patch, and early phosphorylated Ser768 may not attenuate channel activation by prohibiting PKA phosphorylation at some stimulatory sites, as suggested by Csana´dy and co-workers (24). Login to comment 327 ABCC7 p.Ser768Ala Although phosphoserine Ser768 cannot form an H-bond with His950 , the maximal open probability of WT CFTR was found to be lower than that of S768A (24). Login to comment 337 ABCC7 p.Ser768Ala Effects of cAMP on Channel Gating-Previous studies demonstrated that the S768A mutant expressed in Xenopus oocytes exhibited weak phosphorylation of the R domain, high base-line activity, substantial activation by isobutylmethylxanthine/forskolin, and slight inhibition by local AMPK activation (18, 24, 25). Login to comment 340 ABCC7 p.Ser768Asp This difference may not result from phosphorylation of Ser768 because a basal open probability of S768D was higher (Po ϭ 0.0004) than that of WT CFTR (Fig. 9). Login to comment 342 ABCC7 p.His950Ala Supporting this proposal, H950A greatly facilitated basal channel opening in the resting cell, possibly because this mutation increased sensitivity to endogenous ATP and promoted phosphorylation at some stimulatory sites primarily by weakening endogenous Fe3ϩ binding (Fig. 9). Login to comment 343 ABCC7 p.His950Arg ABCC7 p.His950Arg ABCC7 p.Ser768Arg It is expected that H950R and H950R/S768R may also exert similar effects on the basal channel opening. Login to comment 344 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp ABCC7 p.His950Ala Unlike H950A, a basal channel open probability of S768A and S768D was still low (Po ϭ 0.0004) (Fig. 9). Login to comment 346 ABCC7 p.Ser768Asp ABCC7 p.His950Asp A similar observation would be seen with H950D/S768D. Login to comment 347 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp ABCC7 p.His950Ala Despite this complex involvement, H950A, S768A, and S768D were more sensitive to forskolin than WT CFTR because they were dramatically activated soon after forskolin was introduced (Fig. 9). Login to comment