ABCMdb

ABCC7 p.Ser768Ala

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PMID: 14602047 [PubMed] Howell LD et al: "Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain."

No. Sentence Comment

84 Similarly, the substitution of Ala for Ser-768 resulted in the loss of phosphopeptides 2, 3 and 8, indicating that these three peptides contain Ser-768 (Figure 1E). Login to comment

PMID: 15155835 [PubMed] Ai T et al: "Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents."

No. Sentence Comment

142 We further explored the action of capsaicin using CFTR mutants whose eight major PKA consensus serines are substituted with alanine (S660A, S686A, S700A, S712A, S737A, S768A, S795A, and S813A), so called S-oct-A or 8SA. Login to comment

PMID: 15657296 [PubMed] Csanady L et al: "Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA."

No. Sentence Comment

4 The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. Login to comment
5 In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. Login to comment
6 As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. Login to comment
7 The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. Login to comment
8 The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. Login to comment
41 One consequence of this effect of phosphoserine 768 is that the dose-response curve for activation of CFTR chloride current by PKA is shifted to higher [PKA] for WT channels than for mutant S768A CFTR channels. Login to comment
44 S768A CFTR cDNA, provided by D. Dawson (OHSU, Portland, OR), was sub- cloned into the SmaI and XhoI sites of pGEMHE. Login to comment
71 To allow comparison of activation rates of WT and S768A CFTR channels, current relaxation time courses were fitted with single exponentials by nonlinear least squares (SigmaPlot 7.0). Login to comment
121 Large Resting Conductance of Xenopus Oocytes Expressing Mutant S768A CFTR We compared the activity of WT and Ser-768-to-Ala mutant CFTR channels after expressing them in oocytes. Login to comment
135 Two-microelectrode voltage-clamp recordings revealed measurable membrane conductance in resting oocytes expressing WT or S768A CFTR (Fig. 3, B-D, plot c and e). Login to comment
137 RpcAMPS injection reduced basal WT CFTR conductance from 12 Ϯ 2 to 4 Ϯ 1 ␮S (n ϭ 8), and similarly lowered the much larger basal S768A CFTR conductance from 146 Ϯ 9 (n ϭ 9) to 7 Ϯ 2 ␮S (n ϭ 5; e.g., Fig. 3 E). Login to comment
138 Stimulation of the endogenous cAMP-PKA pathway by superfusion with 1 mM IBMX ϩ 50 ␮M forskolin robustly increased membrane conductance in WT CFTR-injected oocytes (to 151 Ϯ 5 ␮S, n ϭ 10; Fig. 3, B and D, plot d), but only slightly increased membrane conductance in oocytes expressing S768A CFTR (to 178 Ϯ 4 ␮S, n ϭ 9; Fig. 3, C and D, plot f). Login to comment
139 Thus, although the maximally activated conductances of oocytes expressing S768A or WT CFTR were comparable, the ratios of their resting conductance to maximally activated conductance were very different (Fig. 3 F), averaging 0.16 Ϯ 0.02, n ϭ 10, for WT but 0.82 Ϯ 0.04, n ϭ 9, for S768A. Login to comment
140 Because the maximum conductance (‫081ف‬ ␮S) activated by forskolin ϩ IBMX in oocytes injected with Ն2.5 ng CFTR cRNA possibly reflects saturation of some component in the oocyte cAMP-PKA pathway (Csanády et al., 2000), further comparison of S768A and WT channel activity was limited to excised patches (see Figs. 5-7, below). Login to comment
141 Insofar as the Ser to Ala mutation at position 768 may be expected to little alter the structure, and hence the function, of CFTR channels in the absence of phosphorylation, the large difference between the activation levels of S768A and WT CFTR channels in resting oocytes suggests that the basal PKA activity in those oocytes was sufficient to phosphorylate Ser 768 in WT CFTR, which then exerted its inhibitory influence on CFTR conductance. Login to comment
142 Moreover, that limited conductance of WT CFTR, as well as the substantial conductance of S768A CFTR, in resting oocytes suggests that the basal activity of PKA was also able to sustain steady-state phosphorylation of at least one stimulatory site in CFTR. Login to comment
145 Membrane conductance of resting and activated oocytes expressing WT or mutant S768A CFTR. Login to comment
146 Current time courses, recorded under two-microelectrode voltage clamp, of oocytes injected with water (A), or with cRNA encoding (B) WT or (C) S768A CFTR. Login to comment
150 (E) Rapid reduction of resting conductance (from 145 to 8 ␮S) upon injection of Rp-cAMPS into an oocyte expressing S768A CFTR. Login to comment
151 (F) Ratios, for WTand S768A-expressing oocytes, of membrane conductances at rest (Grest), and after maximal activation of CFTR (Gmax); conductance values were similar for oocytes injected with 2.5 or 5 ng of each cRNA, so pooled results are shown. Login to comment
168 high basal activity of S768A CFTR channels can reasonably be attributed to absence of that phosphorylation and, hence, lack of its inhibitory effect on CFTR current. Login to comment
169 By the same token, our finding that serines 660, 700, 712, 737, and 795 were also phosphorylated means that these are candidates for the stimulatory site (or sites) underlying the observed activity of WT and S768A CFTR channels in resting oocytes. Login to comment
170 Fractional Activation by Low [PKA] is Enhanced for S768A Mutant Channels We examined the inhibitory influence of phosphoserine 768 by comparing channel function in inside-out patches excised from oocytes expressing WT (Fig. 5 A) or S768A mutant (Fig. 5 B) CFTR. Login to comment
171 Though the patches contained hundreds of channels, no appreciable currents flowed in either WT or S768A channels when their cytoplasmic surfaces were exposed to 2 mM MgATP, ‫2ف‬ min after patch excision. Login to comment
172 But both WT and S768A channels were activated when first a low (55 nM), and then a high (550 nM), concentration of PKA catalytic subunit was added to the MgATP. Login to comment
173 The fractional current activated by the low [PKA], relative to that subsequently elicited by 550 nM PKA, was evidently larger for S768A (Fig. 5, B and C; 0.57 Ϯ 0.06, n ϭ 5) than for WT channels (Fig. 5, A and C; 0.35 Ϯ 0.03, n ϭ 7). Login to comment
174 For both WT and S768A channels, 550 nM PKA was almost a saturating concentration, since fractional activation was already high at 220 nM PKA (I220/I550 values were 0.80 Ϯ 0.03, n ϭ 3 for WT, 0.86 Ϯ 0.09, n ϭ 3 for S768A). Login to comment
175 At low [PKA], S768A channels were also activated more rapidly than WT CFTR channels, and after a shorter delay (Fig. 5, A and B). Login to comment
177 The resulting time constants for WT (gray bars) and S768A (black bars) are summarized in Fig. 5 D. Login to comment
178 On exposure to 55 nM PKA, the current increase was twice as fast, on average, for S768A (␶relax ϭ 25 Ϯ 5 s, n ϭ 5) as for WT CFTR (␶relax ϭ 56 Ϯ 12 s, n ϭ 5; Fig. 5 D, top). Login to comment
181 Activation in excised patches of macroscopic WT and S768A CFTR currents by low and high concentrations of PKA catalytic subunit. Login to comment
182 (A and B) Currents recorded in patches containing hundreds of WT (A) or S768A (B) CFTR channels. Login to comment
185 (C) Fractional current activated by 55 nM PKA was significantly smaller for WT (gray bar) than S768A (black bar; *, P ϭ 0.0024) CFTR channels; mean steady current in 55 nM PKA was divided by mean steady current in the same patch at 550 nM PKA. Login to comment
186 (D) Time constants of macroscopic current relaxations upon addition of 55 nM (top) or 550 nM (bottom) PKA, for WT (gray bars) and S768A (black bars) CFTR; activation was faster for S768A at low (*, P ϭ 0.036), but not at high [PKA]. Login to comment
187 Longer Burst Durations Underlie Higher Open Probability of S768A Channels Channel gating characteristics underlying the differences in macroscopic currents were investigated in excised patches with fewer channels; currents from patches containing four WT and five S768A channels are shown in Fig. 6, A and B, respectively. Login to comment
190 The fractional increase in Po on increasing [PKA] from 55 to 550 nM replicated the observed macroscopic current ratios (approximately threefold for WT, Fig. 6 C, gray bars, Ͻ2-fold for S768A, Fig. 6 C, black bars; c.f. Fig. 5 C). Login to comment
191 However, absolute Po of S768A channels was at least 50% higher than that of WT at 550 nM PKA (0.48 Ϯ 0.05, n ϭ 6 for S768A vs. 0.29 Ϯ 0.02, n ϭ 9 for WT) and approximately threefold higher at 55 nM PKA (0.25 Ϯ 0.03, n ϭ 4 for S768A vs. 0.07 Ϯ 0.01, n ϭ 4 for WT; Fig. 6 C). Login to comment
192 At 55 nM PKA, interburst durations were somewhat shorter for S768A (2681 Ϯ 534 ms, n ϭ 4; Fig. 6 D, black bar) than for WT (3805 Ϯ 454 ms, n ϭ 4; Fig. 6 D, gray bar), a difference not apparent at 550 nM PKA (824 Ϯ 62 ms, n ϭ 6 for S768A vs. 958 Ϯ 71 ms, n ϭ 9 for WT; Fig. 6 D). Login to comment
193 Open burst durations of S768A channels were Ն2-fold longer (Fig. 6 E, black bars) than those of WT channels (Fig. 6 E, gray bars) both at 55 nM (941 Ϯ 282 ms, n ϭ 4 vs. 335 Ϯ 49 ms, n ϭ 6) and at 550 nM PKA (1036 Ϯ 376 ms, n ϭ 6 vs. 435 Ϯ 38 ms, n ϭ 10). Login to comment
194 Interestingly, the open burst duration of S768A channels was reduced at least threefold (to ␶b ϭ 234 Ϯ 28 ms, n ϭ 5) when measured shortly after withdrawal of PKA (see Fig. S1, available at http://www.jgp.org/cgi/ content/full/jgp200409076/DC1), just as we have previously reported for WT CFTR channels (Csanády et al., 2000; Vergani et al., 2003). Login to comment
195 Also as we have found for WT CFTR, macroscopic current in excised patches containing phosphorylated S768A channels (just after PKA removal) was half-maximally activated by roughly 50 ␮M MgATP (Michaelis fit yielded K0.5 ϭ 40 Ϯ 2 ␮M MgATP; see Fig. S2, available at http://www.jgp.org/ cgi/content/full/jgp200409076/DC1). Login to comment
196 The enhanced fractional activation at low [PKA] of S768A channels relative to WT CFTR channels, evident in comparisons of amplitudes of macroscopic current (Fig. 5 C) or of Po (Fig. 6 C), implies that the mutant channels display a higher apparent "affinity" for PKA (at least, as assayed by channel activity). Login to comment
198 Kinetic behavior of WT and S768A CFTR channels in excised patches exposed to low and high [PKA]. Login to comment
199 Representative baseline-subtracted current traces of (A) four WT and (B) five S768A channels, recorded from excised patches in the presence of 2 mM MgATP ϩ 55 nM or 550 nM PKA; 20-s segments (indicated by bars) under each condition are shown with 10-fold expanded time scale, below. Login to comment
201 (C-E) Open probabilities (C), mean interburst (D) and open burst (E) durations at 55 nM (top) or 550 nM PKA (bottom), for WT (gray bars) and S768A (black bars) CFTR channels; asterisks indicate significant differences between S768A and WT (0.001 Ͻ P Ͻ 0.06). Login to comment
202 against [PKA] for S768A and WT channels (Fig. 7). Login to comment
203 Half-maximal activation of Po requires ‫051ف‬ nM PKA for WT channels, but only ‫07ف‬ nM PKA for S768A channels. Login to comment
204 The Hill coefficient was 1.5 for WT and 1.8 for S768A, suggesting that more than one site on a CFTR channel must be phosphorylated before its Po becomes measurable; this is also consistent with the sigmoid time courses of current activation observed at low [PKA] (Fig. 5, A and B). Login to comment
205 Comparable Phosphorylation Time Courses of Recombinant WT and S768A R-domain Peptides Our mass spectrometric and functional analysis of WT CFTR expressed in oocytes (Figs. 3 and 4) revealed in vivo phosphorylation of serines involved in channel activation as well as of the inhibitory Ser 768. Login to comment
207 We addressed this possibility by examining the phosphorylation kinetics of His-tagged WT and mutant S768A R-domain protein incubated with PKA and ␥32P-MgATP to see whether differences were discernible. Login to comment
208 In addition, to verify the inferred link between the major mobility shift of the R domain and phosphorylation of Ser 737 (Fig. 2), the other candidate inhibitory serine (Wilkinson et al., 1997), we studied phosphorylation of His-tagged R-domain proteins containing the single mutation S737A, or the double mutation S737A-S768A. Login to comment
209 The incremental mobility shifts already seen in Figs. 1 and 2 as phosphorylation of the R domain progressed were recapitulated in the His-tagged WT peptide (Fig. 8 A), and were not substantially altered by the S768A mutation in either the WT or S737A background (Fig. 8, B and D vs. A and C). Login to comment
210 As anticipated, however, the S737A mutation abolished the large mobility shift of both WT and S768A R-domain peptides (Fig. 8, C and D vs. A and B). Login to comment
212 Dependence on [PKA] of open probability, Po, of WT (᭹) and S768A (᭺) CFTR channels. Login to comment
214 Lines show nonlinear least-squares fits to the Hill equation, yielding Po,max ϭ 0.34 Ϯ 0.06, K0.5 ϭ 149 Ϯ 46 nM, nH ϭ 1.5 Ϯ 0.5 for WT, and Po,max ϭ 0.51 Ϯ 0.05, K0.5 ϭ 71 Ϯ 12 nM, nH ϭ 1.8 Ϯ 0.5 for S768A. Login to comment
217 WT (A) and mutant R-domain peptides, with Ser768 replaced by alanine (S768A; B), Ser737 replaced by alanine (S737A; C), or Ser737 and Ser768 both replaced by alanine (S737A-S768A; D), were phosphorylated with 10 nM PKA and 5 ␮M ␥32P-MgATP for 0.5-60 min as indicated. Login to comment
219 The major mobility shift (to band 3; Figs. 1 and 2) was seen in the WT and S768A peptides, but not in the S737A or S737A-S768A peptides. Login to comment
220 The kinetics of R-domain phosphorylation, as demonstrated by the mobility shifts, was little altered in the two S768A mutants. Login to comment
221 (E and F) Two-dimensional tryptic phosphopeptide maps of His-tagged WT and S768A R-domain peptides phosphorylated in vitro as in A and B, but for 0.5 min with 10 nM PKA and 50 ␮M ␥32P-MgATP, and then subjected to SDS-PAGE and analyzed by autoradiography. Login to comment
224 Four spots (arrows) in the WT R domain map are absent from the S768A map, but no similarly striking differences are seen in the pattern or intensity of other spots. Login to comment
226 To more closely investigate the possibility that phosphoserine 768 impairs phosphorylation of specific sites, 2-D phosphopeptide maps were prepared of WT and S768A R-domain peptides phosphorylated in vitro as in Fig. 8, A and B. Login to comment
227 If phosphoserine 768 inhibits or delays phosphorylation of other serines, then its absence from the mutant S768A R domain might result in new spots, or spots with higher intensity, in the S768A map. Login to comment
228 On the contrary, however, the principal difference between the two phosphopeptide maps of WT and S768A R-domain samples obtained from lower gel bands, after 30 s of phosphorylation in the presence of 50 ␮M MgATP, is the omission from the S768A map of four spots in the WT map (arrows, Fig. 8, E and F) that can therefore be presumed to contain phosphoserine 768. Login to comment
229 In particular, the strongest spots in the phosphopeptide map of phosphorylated S768A R domain are also seen in the WT map; the S768A map contains no obvious novel or intensified spots, and so provides no clear evidence for major enhancement of phosphorylation of other R-domain serines. Login to comment
234 We found that mutation of Ser 768 to alanine enhanced average CFTR current in excised patches at all levels of [PKA], but especially at low [PKA], confirming the inhibitory role of Ser 768 proposed earlier (Wilkinson et al., 1997). Login to comment
258 The increase in sensitivity was largest (approximately fivefold) for S768A mutant CFTR (Wilkinson et al., 1997). Login to comment
259 We confirm that inhibitory influence of phosphoserine 768 here by directly showing in excised patches that the sensitivity to activation by PKA catalytic subunit is shifted to lower [PKA] for S768A channels compared with WT CFTR channels (Figs. 5-7). Login to comment
260 Accordingly, we found that the basal level of [PKA] (expected to be low) in resting oocytes caused a greater activation of S768A channels than of WT channels (Fig. 3). Login to comment
261 Strictly, in the absence of structural information, we cannot rule out the possibility that the enhanced sensitivity of S768A channels results not from loss of an inhibitory PKA phosphorylation site but from a structural change caused by the Ser-Ala mutation itself. Login to comment
265 If that assumption is correct, a corollary of our observation of a larger chloride conductance in unstimulated oocytes expressing S768A channels than in those expressing WT CFTR (Fig. 3) is that Ser 768 should be phosphorylated in WT channels in resting oocytes. Login to comment
267 We can be sure that the large conductance observed for S768A mutant CFTR in resting oocytes (Fig. 3) was phosphorylation dependent, and did not reflect constitutive channel activity induced simply as a consequence of the Ser-Ala mutation itself, because the conductance was abolished by injection of the PKA inhibitor Rp-cAMPS. Login to comment
268 That high resting conductance of S768A channels thus depended on continuous phosphorylation of stimulatory sites by the relatively low PKA activity in the unstimulated oocytes, consistent with the enhanced sensitivity of S768A channels to PKA we found in excised patches (Fig. 7). Login to comment
269 The deactivation of the S768A channels upon kinase inhibition by RpcAMPS means that phosphatases must also have been continuously active in the resting oocytes. Login to comment
270 In support of this interpretation, 2 mM MgATP caused negligible opening of S768A channels in excised patches, ‫2ف‬ min after excision, before application of exogenous PKA, just as found for WT channels (Fig. 5, A and B), suggesting that, even in the excised patch, membrane-associated phosphatases dephosphorylate at least the stimulatory sites and so deactivate both WT and S768A CFTR channels (compare Chan et al., 2000; Csanády et al., 2000). Login to comment
272 Direct comparison of the gating kinetics of S768A and WT CFTR in excised patches showed that the Po of S768A channels was greater than that of WT channels at all [PKA] tested, and was at least 50% greater at saturating, 550 nM, PKA (Fig. 7), largely because the open burst duration of S768A channels was roughly double that of WT channels at 550 nM PKA. Login to comment
278 In that case, the observed ‫%05ف‬ increase in Po,max of S768A channels (Fig. 7) alone would suffice to reduce the half-maximally activating [PKA], KP, by about one third compared with that for WT channels, without any influence of phosphoserine 768 on phosphorylation of other sites (and hence on Kr). Login to comment
279 However, the measured change in the apparent affinity for Po activation by PKA caused by the S768A mutation appeared larger than that, around twofold (Fig. 7), and so an additional indirect influence of this mutation on channel phosphorylation cannot be ruled out. Login to comment
282 Nor could we find in 2-D phosphopeptide maps any peptides phosphorylated to obviously higher stoichiometry in S768A than in WT R domain at early times during the phosphorylation time course (Fig. 8, E and F). Login to comment
286 Our finding that, at low [PKA], the somewhat sigmoid macroscopic current activation was slower for WT than for S768A channels and occurred after a longer delay (Fig. 5) is consistent with an inhibitory influence of phosphoserine 768 on subsequent phosphorylation of other, activating, sites in WT CFTR. Login to comment
287 However, the left-shifted Po vs. [PKA] curve of S768A channels (Fig. 7) could by itself provide an explanation for their faster current activation, even if phosphorylation of all other serines were unaffected by the S768A mutation. Login to comment
288 In that case, for both WT and S768A channels, the [PKA] axis of the steady-state Po vs. [PKA] curves (Fig. 7) could be rescaled to read the same steady level of phosphorylation. Login to comment
289 Then, by hypothesis, on exposure to a given [PKA] the time course of phosphorylation would be the same for WT and S768A channels, equivalent to moving to the right at the same speed along the now rescaled abscissa of Fig. 7. Login to comment
290 This would result in a faster climb of the ordinate for S768A CFTR, and hence faster current rise, as observed (Fig. 5). Login to comment
291 In summary, we have demonstrated a direct influence of phosphoserine 768 to speed closure from bursts in WT CFTR channels which, in principle, offers at least a qualitative explanation for every other effect we observe, including their reduced sensitivity to activation by PKA and slower activation time course, relative to S768A CFTR. Login to comment
296 Mass spectrometry and site-directed mutagenesis revealed that the major mobility shift was linked to phosphorylation of Ser 737 both at low and high [MgATP] (Fig. 2), and this requirement was confirmed by the absence of the large mobility shift after mutation of Ser 737 to Ala in either WT or S768A R-domain peptide (Fig. 8, A-D; see also Borchardt et al., 1996; Kole et al., 1998). Login to comment
298 In contrast, phosphorylation of Ser 768 was accompanied by no discernible shift in R-domain mobility (Fig. 2), and mutating Ser 768 to Ala did not obviously alter the pattern of mobility shifts upon phosphorylation of the R domain (Fig. 8, A-D). Login to comment
299 Though we did not examine its consequences for channel gating, the S737A mutation has been reported to leave burst duration unchanged from that of WT CFTR (Winter and Welsh, 1997), whereas we found the S768A mutation to roughly double burst duration (Fig. 6). Login to comment
308 Ser 768 cannot be the responsible site because the burst duration of S768A CFTR channels, like that of WT, was reduced at least twofold following withdrawal of PKA (Fig. S1). Login to comment
320 The ready phosphorylation of Ser 768 in WT CFTR channels results in a somewhat greater reduction of Po at low than at high [PKA], so shifting the dose-response curve for WT to the right relative to that for S768A CFTR channels. Login to comment

PMID: 17700961 [PubMed] Quinton PM et al: "Too much salt, too little soda: cystic fibrosis."

No. Sentence Comment

78 Substitutionofalanine for phosphorylatable S737A or S768A enhanced the channel activity, suggesting that phosphorylation at either of these sites may inhibit phosphorylation of other stimulatory sites[72] . Login to comment

PMID: 19095655 [PubMed] Kongsuphol P et al: "Mechanistic insight into control of CFTR by AMPK."

No. Sentence Comment

43 EXPERIMENTAL PROCEDURES cRNAs for CFTR and Double Electrode Voltage Clamp-Oocytes were injected with cRNA (10 ng, 47 nl of double-distilled water) encoding wtCFTR, L1430A/L1431A, S573A, S1248A, F508del-CFTR, G551D-CFTR, S768A, S737A, S768D, S737D, E1474X, and AMPK␣1. Login to comment
70 The S768A mutant of CFTR abrogated almost entirely by phosphorylation of AMPK. Login to comment
93 Fig. 2B demonstrates that AMPK indeed phosphorylates the R domain in vitro and this AMPK phosphorylation is largely reduced in R domain mutants S737A and S768A (compare lanes 2 and 3 in Fig. 2B, lower panel). Login to comment
94 The data also suggest that serine 768 has a much greater reductive impact because S768A almost abolished all the phosphorylation, whereas some were preserved with S737A. Login to comment
95 Crucially, the double mutant labeling was similar to S768A alone. Login to comment
96 We quantified data (supplemental Fig. S2) from three independent experiments and found that the S737A mutation leads to a small 23 Ϯ 8% reduction in counts, whereas S768A leads to a major 81 Ϯ 5% (mean Ϯ range) reduction similar to the double mutant (87% Ϯ 4%) when compared with the wild type (100%). Login to comment
125 It is clear that S768A (lower left panel) has a more profound inhibitory effect on R domain phosphorylation compared with S737A (upper right) given that all these experiments were run simultaneously with similar concentrations of R domain protein and kinase, and each imaged for identical lengths of time. Login to comment
126 Broadly, prolonged incubation that previously created two major spots found with AMPK alone was now supplemented by multiple small spots that almost disappeared after S768A mutation, but much less so after S737A mutation (Fig. 2D, compare lower two panels with upper). Login to comment
128 When expressed in oocytes, CFTR bearing the R domain mutants S737A and S768A as well as the double mutant S737A/ S768A (Fig. 3A) produced dramatically enhanced conductances (S768A Ͼ S737A; Fig. 3B) upon stimulation with forskolin (2 ␮M) and IBMX (1 mM). Login to comment
130 The large Cl-conductances generated by S737A/S768A were inhibited by 5 ␮M of the specific chloride channel blocker CFTRinh-172 (Fig. 3D), or alternatively, the PKA inhibitor KT5720 and Cl- replacement by gluconate (not shown). Login to comment
131 Once again the differential roles of these two serines were observed because the conductance observed with S737A was almost 100 ␮S lower than that found with S768A. Login to comment
135 B, phosphoryl- ationoftheRdomainwithPKAandAMPK.AMPKphosphorylationwaslargely reduced or abolished in S737A and S768A, respectively (lower panel). Login to comment
143 The S768A mutant abrogates almost all the phosphorylation. Login to comment
145 Regulation of CFTR by AMPK 5648 respectively) with the S573A but not with the S768A (compare second and third panels of Fig. 3C with the corresponding wild type result in the first panel of the figure). Login to comment
146 Overall the data suggest that S737A, S768A, and the double mutant S737A/ S768A were no longer sensitive to stimulation or inhibition of AMPK indicating that phosphorylation at both serines is required (Fig. 3C, middle panels). Login to comment
151 In contrast to wtCFTR the CFTR mutants S737A (not shown), S768A, and S737A/S768A produced a high Cl- con- ductance under basal conditions, i.e. in the absence of IBMX and forskolin (Fig. 4, A and B). Login to comment
155 A, whole cell currents activated by IBMX (1 mM) and forskolin (2 ␮M) in wtCFTR and S737A/ S768A-CFTRexpressingoocytes.B,summaryofwholecellconductancesgen- erated by wtCFTR and different CFTR mutants. Login to comment
156 C, summary of CFTR whole cell conductances generated by wtCFTR and different CFTR mutants, and effects of phenformin and compound C. D, summary of the whole cell conductance activated by S737A/S768A-CFTR and inhibition by the CFTR blocker CFTRinh-172. Login to comment
161 S737A/S768A-CFTR generates a baseline conductance. Login to comment
162 A, effect of extracellular Cl- replacement by gluconate (glcn) on whole cell currents generated by wtCFTR, S768A-CFTR, and S737A/S768A-CFTR in the absence of IBMX and forskolin. Login to comment
164 C, summary of the whole cell conductances generated by wtCFTR and S737A/S768A-CFTRintheabsenceofstimulationwithIBMXandforskolinand effectsofphenformin,compoundC,andCFTRinh-172.DandE,baselinewhole cell conductances generated by wild type CFTR (D) and S737A/S768A-CFTR (E) and effects of compound C and the PKA inhibitor KT520. Login to comment
170 Moreover, the PKA inhibitor KT5720 (50 ␮M) inhibited this enhanced baseline CFTR conductance generated by S737A/ S768A irrespective of the presence of compound C (Fig. 4E). Login to comment
172 We interpret this finding to suggest that the sensitivity of S737A/ S768A-CFTR toward PKA inhibition is retained, which implies that PKA is now active after the loss of AMPK sensitivity. Login to comment
178 The initial recovery from PKA stimulation was equal in S737A/ S768A (1.1 Ϯ 0.3 ␮S/min) and wtCFTR (1.1 Ϯ 0.1 ␮S/min) (Fig. 5C), but was enhanced in ooctyes coexpressing kinase-dead AMPK␣1-K45R (2.8 Ϯ 0.4 ␮S/min), whereas overexpression of wtAMPK␣1beta1␥1 literally eliminated CFTR currents (Fig. 5B). Login to comment
180 Although AMPK largely antagonizes activation of CFTR, the mutated serines S737A and S768A do not seem to influence the recovery time from forskolin stimulation. Login to comment
210 C, inactivation of conductances generated by wtCFTR and S737A/S768A-CFTR. Login to comment
225 Even maximal stimulation of wtCFTR with a mixture of 8-Br- - cAMP, IBMX, and forskolin does not produce the same high level of conductance as S737A-CFTR or S768A-CFTR. Login to comment

PMID: 19419994 [PubMed] King JD Jr et al: "AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR."

No. Sentence Comment

79 One day after harvesting, oocytes were injected with either wild-type CFTR or CFTR-S768A cRNA (1 ng). Login to comment
140 AMPK-dependent phosphorylation of CFTR-S768A was substantially reduced (by ϳ70%) compared with that of wild-type CFTR, to levels similar to that of the CFTR-10SA mutant (Fig. 4, B and C). Login to comment
144 It was reported previously that CFTR-S768A exhibits a very high open probability, whereas the phospho-mimic Ser-to-Asp S768D CFTR mutant has a very low open probability relative to wild-type CFTR following PKA stimulation (8, 27, 28). Login to comment
147 The relevance of Ser768 for AMPK modulation of CFTR activity was further investigated in TEV studies of Xenopus oocytes expressing either wild-type CFTR or CFTR-S768A. Login to comment
151 Peak currents following stimulation with PKA agonists were significantly greater in oocytes expressing CFTR-S768A than in those expressing CFTR-WT, consistent with previous results (8). Login to comment
152 Of note, ZMP was without effect on the peak conductance of oocytes expressing CFTR-S768A, suggesting that this mutant is resistant to Fig. 4. Login to comment
157 The WT and S768A images shown are from a different membrane than the rest of the mutants shown. Login to comment
158 C: in vitro phosphorylation, corrected for CFTR protein expression and normalized to that of CFTR-WT (*P Ͻ 0.01, unpaired t-tests compared with the S768A, 10SA, and 10SA-A737S mutants; n ϭ 4-6 separate experiments). Login to comment
159 There were no significant differences in phosphorylation intensity among the S768A, 10SA, and 10SA-A737S mutants (P Ͼ 0.10, unpaired t-tests). Login to comment
163 In addition, baseline, unstimulated CFTR conductances were enhanced by approximately fivefold in oocytes expressing CFTR-S768A (Fig. 5C), suggesting that CFTR-WT conductance is tonically inhibited in oocytes by Ser768 phosphorylation. Login to comment
174 A: representative whole cell conductance recordings from control [potassium gluconate (KG)]-injected oocytes expressing either CFTR-WT or CFTR-S768A. Login to comment
176 B: mean (Ϯ SE) peak conductances normalized to the mean control (KG)-injected, CFTR-WT peak conductance for that experimental day and batch in CFTR-WT versus CFTR-S768A-expressing oocytes injected with either control (KG) or AMPK activator [5-aminoimidazole-4-carboxamide ribonucleoside monophosphate (ZMP)]. Login to comment
177 A significant decrease in relative peak conductance was observed for ZMP-injected, CFTR-WT-expressing oocytes (*P Ͻ 0.01) but not for ZMP-injected CFTR-S768A-expressing oocytes. Login to comment
179 CFTR-S768A-expressing oocytes had a dramatically elevated prestimulation starting conductance relative to that of CFTRWT-expressing oocytes. Login to comment
180 There was also a ϳ40-50% decrease in starting conductance of CFTR-WT-expressing oocytes injected with ZMP versus KG (#P Ͻ 0.05), whereas there was no difference in starting conductance between the two conditions for CFTR-S768A-expressing oocytes. Login to comment
182 Whole cell CFTR conductance before and after addition of PKA agonists with or without AMPK activation in Xenopus oocytes Starting Conductance, ␮S Peak Conductance, ␮S CFTR-WT ϩ KG 12.4Ϯ1.4 67.0Ϯ9.3 CFTR-WT ϩ ZMP 8.5Ϯ0.9 30.5Ϯ3.8 CFTR-S768A ϩ KG 146.9Ϯ13.0 256.6Ϯ15.6 CFTR-S768A ϩ ZMP 128.5Ϯ9.0 238.7Ϯ13.7 Values are means Ϯ SE of whole cell two-electrode voltage clamp conductances measured before and at the peak after infusion of 1 ␮M forskolin and 100 ␮M IBMX. Login to comment
190 Consistent with previous observations (8), we found that mutation of this site to Ala (S768A) greatly enhanced baseline, unstimulated conductance of CFTR expressed in Xenopus oocytes, and it reduced the relative activation of CFTR by PKA agonists (Fig. 5). Login to comment
191 The apparent constitutive activation of CFTR-S768A is reminiscent of that observed for CFTR-WT in cells with AMPK activity inhibited by overexpression of AMPK-DN (Fig. 1). Login to comment
193 The importance of AMPK phosphorylation at Ser768 is further demonstrated by the observation that whereas PKA stimulation of CFTR-WT conductance was inhibited by the AMPK activator ZMP, PKA stimulation of CFTR-S768A was insensitive to ZMP. Login to comment
198 Indeed, a residual AMPK-mediated phosphorylation of 25-30% was observed in CFTR-S768A (Fig. 4), suggesting that other AMPK phosphorylation sites in CFTR may exist. Login to comment
78 One day after harvesting, oocytes were injected with either wild-type CFTR or CFTR-S768A cRNA (1 ng). Login to comment
139 AMPK-dependent phosphorylation of CFTR-S768A was substantially reduced (by ϳ70%) compared with that of wild-type CFTR, to levels similar to that of the CFTR-10SA mutant (Fig. 4, B and C). Login to comment
143 It was reported previously that CFTR-S768A exhibits a very high open probability, whereas the phospho-mimic Ser-to-Asp S768D CFTR mutant has a very low open probability relative to wild-type CFTR following PKA stimulation (8, 27, 28). Login to comment
146 The relevance of Ser768 for AMPK modulation of CFTR activity was further investigated in TEV studies of Xenopus oocytes expressing either wild-type CFTR or CFTR-S768A. Login to comment
150 Peak currents following stimulation with PKA agonists were significantly greater in oocytes expressing CFTR-S768A than in those expressing CFTR-WT, consistent with previous results (8). Login to comment
156 The WT and S768A images shown are from a different membrane than the rest of the mutants shown. Login to comment
162 In addition, baseline, unstimulated CFTR conductances were enhanced by approximately fivefold in oocytes expressing CFTR-S768A (Fig. 5C), suggesting that CFTR-WT conductance is tonically inhibited in oocytes by Ser768 phosphorylation. Login to comment
173 A: representative whole cell conductance recordings from control [potassium gluconate (KG)]-injected oocytes expressing either CFTR-WT or CFTR-S768A. Login to comment
175 B: mean (Ϯ SE) peak conductances normalized to the mean control (KG)-injected, CFTR-WT peak conductance for that experimental day and batch in CFTR-WT versus CFTR-S768A-expressing oocytes injected with either control (KG) or AMPK activator [5-aminoimidazole-4-carboxamide ribonucleoside monophosphate (ZMP)]. Login to comment
178 CFTR-S768A-expressing oocytes had a dramatically elevated prestimulation starting conductance relative to that of CFTR-WT-expressing oocytes. Login to comment
181 Whole cell CFTR conductance before and after addition of PKA agonists with or without AMPK activation in Xenopus oocytes Starting Conductance, ␮S Peak Conductance, ␮S CFTR-WT ϩ KG 12.4Ϯ1.4 67.0Ϯ9.3 CFTR-WT ϩ ZMP 8.5Ϯ0.9 30.5Ϯ3.8 CFTR-S768A ϩ KG 146.9Ϯ13.0 256.6Ϯ15.6 CFTR-S768A ϩ ZMP 128.5Ϯ9.0 238.7Ϯ13.7 Values are means Ϯ SE of whole cell two-electrode voltage clamp conductances measured before and at the peak after infusion of 1 ␮M forskolin and 100 ␮M IBMX. Login to comment
189 Consistent with previous observations (8), we found that mutation of this site to Ala (S768A) greatly enhanced baseline, unstimulated conductance of CFTR expressed in Xenopus oocytes, and it reduced the relative activation of CFTR by PKA agonists (Fig. 5). Login to comment
192 The importance of AMPK phosphorylation at Ser768 is further demonstrated by the observation that whereas PKA stimulation of CFTR-WT conductance was inhibited by the AMPK activator ZMP, PKA stimulation of CFTR-S768A was insensitive to ZMP. Login to comment
197 Indeed, a residual AMPK-mediated phosphorylation of 25-30% was observed in CFTR-S768A (Fig. 4), suggesting that other AMPK phosphorylation sites in CFTR may exist. Login to comment

PMID: 20952391 [PubMed] Wang G et al: "State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3."

No. Sentence Comment

162 Although Fig. 4E indicates that both S768A and S737A mutants were much inhibited by Fe3ϩ , these sites may not be excluded as Fe3ϩ ligands. Login to comment
165 However, DTT partially reversed Fe3ϩ inhibition of S768A channel activity (Fig. 6B). Login to comment
180 Unlike S768A, S737A exhibited a weak DTT effect (Fig. 6D), suggesting that phosphorylated Ser-737 may not participate in Fe3ϩ binding. Login to comment
237 Macroscopic currents across inside-out membrane patches excised from transfected HEK293T cells expressing the hCFTR (A), S768A (B), and S768D (C) constructs. Login to comment

PMID: 21059651 [PubMed] Wang G et al: "The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

138 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
145 Especially, S768A and S955A, together with CFTR-⌬R, completely removed PKA dependence (Fig. 4F). Login to comment
146 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 It is interesting that both H950A and S768A still needed more PKA to be fully activated in this case (Fig. 5D). Login to comment
167 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
193 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 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 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
213 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
223 However, the activation time became significantly shorter for H950A, S768A, and S768D (Fig. 9, B-E). Login to comment
225 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
234 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
236 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 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 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
246 Furthermore, both S768A and S768D increased sensitivity of CFTR activity to ATP, curcumin, and PKA phosphorylation. Login to comment
263 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 Second, both S768A and S768D were more sensitive to PKA phosphorylation than WT CFTR (Fig. 7D). Login to comment
265 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
272 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 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
293 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
323 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
327 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 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
344 Unlike H950A, a basal channel open probability of S768A and S768D was still low (Po ϭ 0.0004) (Fig. 9). Login to comment
347 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

PMID: 21455600 [PubMed] Siwiak M et al: "Structural models of CFTR-AMPK and CFTR-PKA interactions: R-domain flexibility is a key factor in CFTR regulation."

No. Sentence Comment

121 However, experiments [7] have shown an 80% decrease in AMPK phosphorylation for the CFTR double mutant S737A-S768A. Login to comment
156 This shows that, at the molecular level, it is possible that 20% of the currently unexplained phosphorylation signal observed in the CFTR double mutant S737A-S768A incubated with AMPK may come from "activator" serines such as S813. Login to comment
155 This shows that, at the molecular level, it is possible that 20% of the currently unexplained phosphorylation signal observed in the CFTR double mutant S737A-S768A incubated with AMPK may come from "activator" serines such as S813. Login to comment

PMID: 21594801 [PubMed] Csanady L et al: "Electrophysiological, biochemical, and bioinformatic methods for studying CFTR channel gating and its regulation."

No. Sentence Comment

70 The presence of active endogenous phosphatases in excised patches even permits determination of PKA concentration dependence of CFTR channel activation, assayed as Po; enhanced sensitivity to PKA in Ser768Ala mutants compared to WT CFTR (36) confirmed the inhibitory influence of phosphoserine 768 concluded from measurements of sensitivity to the phosphodiesterase inhibitor IBMX in intact oocytes (46). Login to comment
71 The enhanced sensitivity to phosphorylation resulted in substantial activation of Ser768Ala CFTR channels in resting oocytes, due to basal levels of PKA activity; phosphorylation of six Ser (including Ser768) in the R domains of WT CFTR channels in resting oocytes was confirmed by mass spectrometry (36). Login to comment

PMID: 21594802 [PubMed] Alzamora R et al: "CFTR regulation by phosphorylation."

No. Sentence Comment

92 10SA 10SA-A737S 10SA-A768S A A AI II Phosphorylation Western Blot A I A I WT S768A RelativeInVitroPhosphorylation 0.0 0.2 0.4 0.6 0.8 1.0 1.2 WT S768A 10SA 10SA-A737S 10SA-A768S * Fig. Login to comment
101 The WT and S768A images shown are from a different membrane than the rest of the mutants shown. Login to comment

PMID: 9922377 [PubMed] Gadsby DC et al: "Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis."

No. Sentence Comment

175 In vitro, pre- Po of S768A CFTR channels in excised patches exposed phosphorylation of CFTR by PKC did seem to enhance directly to PKA catalytic subunit suggest that phosphory- subsequent phosphorylation by PKA (31, but see discus- lation of Ser-768 somehow impedes phosphorylation of sion in Ref. 99). Login to comment
183 Nevertheless,site Ser-813 seems to be effectively canceled by phosphorylation of the major inhibitory site Ser-768, since the dou- PKC stimulation or application has invariably been found to potentiate subsequent CFTR channel activation byble mutant S768A/S813A had a K0.5 for IBMX comparable to that of wild-type CFTR channels (220). Login to comment

PMID: 19328185 [PubMed] Hegedus T et al: "Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A."

No. Sentence Comment

132 On the other hand, the much lower rate of S813 phosphorylation appeared to be reduced somewhat further by either the S700A or S737A substitutions, but not by the S768A substitution. Login to comment
152 Membranes isolated from BHK cells expressing S700A (A), S737A (B), S768A (C), or S813A (D) were subjected to phosphorylation for 0, 2, 4, 8, 16, 32, and 64 min. Login to comment
242 The S737A and S768A constructs exhibit slightly higher basal open probability (Po =0.06) compared to wild type (Po ≪0.01) in good agreement with data of Csanady et al. [24]. Login to comment
131 On the other hand, the much lower rate of S813 phosphorylation appeared to be reduced somewhat further by either the S700A or S737A substitutions, but not by the S768A substitution. Login to comment
151 Membranes isolated from BHK cells expressing S700A (A), S737A (B), S768A (C), or S813A (D) were subjected to phosphorylation for 0, 2, 4, 8, 16, 32, and 64 min. Login to comment
241 The S737A and S768A constructs exhibit slightly higher basal open probability (Po =0.06) compared to wild type (Po âa;0.01) in good agreement with data of Csanady et al. [24]. Login to comment

PMID: 18423665 [PubMed] Hegedus T et al: "Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR."

No. Sentence Comment

28 Replacement of serine with alanine at position 737 or 768 was reported to produce a channel with a higher level of activity in both the non-phosphorylated and phosphorylated state.16,19-21 These studies led to the indirect conclusion that these two sites are inhibitory in their phosphorylated state. Login to comment
27 Replacement of serine with alanine at position 737 or 768 was reported to produce a channel with a higher level of activity in both the non-phosphorylated and phosphorylated state.16,19-21 These studies led to the indirect conclusion that these two sites are inhibitory in their phosphorylated state. Login to comment

PMID: 9252549 [PubMed] Wilkinson DJ et al: "CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain."

No. Sentence Comment

87 S686A 0.75 ?I 0.07 9 N PKC site S700A 0.86 t 0.07* 12 S ++ +++ S712A 0.67 t 0.12 9 N - ++ S737A 0.35 5 0.05* 8 I +++ +++ S768A 0.09 IT 0.03* 8 I - ++++ S795A 1.24 + 0.22* 9 S +++ ++++ S813A 3.18-+0.36* 6 S ++++ ++ Values of half-maximal inhibition constant for activation (KA) are means + SE by 3-isobutyl-1-methylxanthine (IBMX) obtained for wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and variants with single serine-to-alanine substitutions. Login to comment
107 Symbols show averaged IBMX dose-response data for wild-type CFTR and several single-site serine-to-alanine mutants (S660A, S737A, S768A, S795A, and S813A). Login to comment
135 CFTR 1O-3 min-l n min 1O-3 mix1 n Wild type 664+51 20 6.0 + 0.3 8826 16 S768A 1,055 5 66* 9 12.7 5 1.7* 112? Login to comment
149 Representative time courses for the activation (A) and deactivation (B) of wild-type (wt) CFTR and single-site mutants S768A and S813A after, respectively, exposure to 10 PM forskolin and 5 mM IBMX and the removal of these drugs from the perfusate. Login to comment
174 It is particularly interesting that S768A, the most important inhibitory site, according to the dose-response assay, has not been clearly identified as an in vivo site. Login to comment

PMID: 7690753 [PubMed] Rich DP et al: "Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain."

No. Sentence Comment

66 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) weretran- siently expressed in HeLa cells. Login to comment
121 The single-channel open-stateprobabil- ity for wild-type CFTR (n = 14), CFTR S-Quad-A (S600A,S737A, S712A,S737A,S768A,S795A,S813A) (n = 7), or CFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D)(n = 7in ATP alone (-PKA);n = 10 inPKA and ATP (+PkX))C1-channels was determined as described under "Experimental Procedures." Login to comment
123 S795A,S813A) (n = 5). Login to comment
174 Fig. 1lA shows that simultaneous substitutionof the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A MockS-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B)or pMT-CFl`R S-Oct-A FIG.9. Login to comment
176 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A)(C). Login to comment
65 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) were transiently expressed in HeLa cells. Login to comment
178 Fig. 1lA shows that simultaneous substitution of the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A Mock S-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B) or pMT-CFl`R S-Oct-A FIG. 9. Login to comment
180 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) (C). Login to comment

PMID: 7684377 [PubMed] Chang XB et al: "Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites."

No. Sentence Comment

37 The following mutations were introduced into CFTR, S422A (TCT to GCT), S660A (TCA to GCA), S686A (TCT to GCT), S700A (TCT toGCT), S712A (TCC to GCC), S737A (TCC to GCC), S768A (TCT toGCT), T788A (ACAto GCA),S795A (TCA to GCA), S813A (TCA to GCA), S660E (TCA to GAA),S737E (TCC to GAG), S795E (TCA to GAA), and S813E (TCA to GAA). Login to comment
45 The DraIII/DraIIIfragment in pNUT-CFTR was replaced by the counterpart from pUCF2.5/6SA to generate 6SA.S686A,S768A, and T788Awere introduced into pUCF2.5/6SA by replacing the counterpart of DraIII/HpaI fragment (containing S660/686/700/712/737/768A) andStyIIStyI fragment (containing T788A/S795/813A). Login to comment

PMID: 23760269 [PubMed] Billet A et al: "Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR)."

No. Sentence Comment

102 Carbachol Stimulates CFTR through PKA and Non-PKA Signaling Pathways-To explore PKA-independent regulation of CFTR without using inhibitors that might have confounding effects on other pathways, we studied the activation of 15SA-CFTR (S422A/S660A/S670A/S686A/T690A/S700A/S712A/ S737A/S753A/S768A/T787A/T788A/S790A/S795A/S813A). Login to comment