ABCMdb

ABCC7 p.Ser768Ala

Predicted by SNAP2:	A: N (61%), C: N (61%), D: N (57%), E: N (61%), F: N (57%), G: N (66%), H: N (61%), I: D (59%), K: N (53%), L: N (53%), M: D (63%), N: N (72%), P: N (53%), Q: N (53%), R: N (53%), T: N (66%), V: N (66%), W: D (66%), Y: D (53%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, T: N, V: D, W: D, Y: D,

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[hide] Protein kinase A regulates ATP hydrolysis and dime... Biochem J. 2004 Feb 15;378(Pt 1):151-9. Howell LD, Borchardt R, Kole J, Kaz AM, Randak C, Cohn JA
Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain.
Biochem J. 2004 Feb 15;378(Pt 1):151-9., 2004-02-15 [PMID:14602047]

Abstract [show]
Gating of the CFTR Cl- channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1-R-GST) on CFTR residues Ser-660, Ser-700, Ser-712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1-R-GST by increasing the apparent K(m) for ATP (from 70 to 250 microM) and the Hill coefficient (from 1 to 1.7) without changing the V(max). When fusion proteins were photolabelled with 8-azido-[alpha-32P]ATP, PKA phosphorylation increased the apparent k(d) for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1-R-GST but not of R-GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2-MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1-R-GST. Thus when the R domain in NBD1-R-GST is phosphorylated by PKA, ATP binding and hydrolysis becomes co-operative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.

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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

[hide] Capsaicin potentiates wild-type and mutant cystic ... Mol Pharmacol. 2004 Jun;65(6):1415-26. Ai T, Bompadre SG, Wang X, Hu S, Li M, Hwang TC
Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.
Mol Pharmacol. 2004 Jun;65(6):1415-26., [PMID:15155835]

Abstract [show]
To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR.

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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

[hide] Preferential phosphorylation of R-domain Serine 76... J Gen Physiol. 2005 Feb;125(2):171-86. Epub 2005 Jan 18. Csanady L, Seto-Young D, Chan KW, Cenciarelli C, Angel BB, Qin J, McLachlin DT, Krutchinsky AN, Chait BT, Nairn AC, Gadsby DC
Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA.
J Gen Physiol. 2005 Feb;125(2):171-86. Epub 2005 Jan 18., [PMID:15657296]

Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. 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. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) 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. 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. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. 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. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

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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

[hide] Too much salt, too little soda: cystic fibrosis. Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415. Quinton PM
Too much salt, too little soda: cystic fibrosis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415., 2007-08-25 [PMID:17700961]

Abstract [show]
Cystic fibrosis (CF) of the pancreas is the most widely accepted name of the most common fatal inherited single gene defect disease among Caucasians. Its incidence among other races is thought to be significantly less, but mutations in the gene have been reported in most, if not all, major populations. This review is intended to give general concepts of the molecular as well as physiological basis of the pathology that develops in the disease. First, an overview of the organ pathology and genetics is presented, followed by the molecular structure of the gene product (cystic fibrosis transmembrane conductance regulator, CFTR), its properties, functions, and controls as currently understood. Second, since mutations appear to be expressed primarily as a defect in electrolyte transport, effects and mechanisms of pathology are presented for two characteristically affected organs where the etiology is best described: the sweat gland, which excretes far too much NaCl ("salt") and the pancreas, which excretes far too little HCO3(- )("soda"). Unfortunately, morbidity and mortality in CF develop principally from refractory airway infections, the basis of which remains controversial. Consequently, we conclude by considering possible mechanisms by which defects in anion transport might predispose the CF lung to chronic infections.

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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

[hide] Mechanistic insight into control of CFTR by AMPK. J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18. Kongsuphol P, Cassidy D, Hieke B, Treharne KJ, Schreiber R, Mehta A, Kunzelmann K
Mechanistic insight into control of CFTR by AMPK.
J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18., 2009-02-27 [PMID:19095655]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser(737) and Ser(768)) in the R domain, formerly identified as "inhibitory" PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser(768) having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.

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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

[hide] AMP-activated protein kinase phosphorylation of th... Am J Physiol Cell Physiol. 2009 Jul;297(1):C94-101. Epub 2009 May 6. King JD Jr, Fitch AC, Lee JK, McCane JE, Mak DO, Foskett JK, Hallows KR
AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR.
Am J Physiol Cell Physiol. 2009 Jul;297(1):C94-101. Epub 2009 May 6., [PMID:19419994]

Abstract [show]
The metabolic sensor AMP-activated protein kinase (AMPK) has emerged as an important link between cellular metabolic status and ion transport activity. We previously found that AMPK binds to and phosphorylates CFTR in vitro and inhibits PKA-dependent stimulation of CFTR channel gating in Calu-3 bronchial serous gland epithelial cells. To further characterize the mechanism of AMPK-dependent regulation of CFTR, whole cell patch-clamp measurements were performed with PKA activation in Calu-3 cells expressing either constitutively active or dominant-negative AMPK mutants (AMPK-CA or AMPK-DN). Baseline CFTR conductance in cells expressing AMPK-DN was substantially greater than controls, suggesting that tonic AMPK activity in these cells inhibits CFTR under basal conditions. Although baseline CFTR conductance in cells expressing AMPK-CA was comparable to that of controls, PKA stimulation of CFTR was completely blocked in AMPK-CA-expressing cells, suggesting that AMPK activation renders CFTR resistant to PKA activation in vivo. Phosphorylation studies of CFTR in human embryonic kidney-293 cells using tetracycline-inducible expression of AMPK-DN demonstrated AMPK-dependent phosphorylation of CFTR in vivo. However, AMPK activity modulation had no effect on CFTR in vivo phosphorylation in response to graded doses of PKA or PKC agonists. Thus, AMPK-dependent CFTR phosphorylation renders the channel resistant to activation by PKA and PKC without preventing phosphorylation by these kinases. We found that Ser768, a CFTR R domain residue considered to be an inhibitory PKA site, is the dominant site of AMPK phosphorylation in vitro. Ser-to-Ala mutation at this site enhanced baseline CFTR activity and rendered CFTR resistant to inhibition by AMPK, suggesting that AMPK phosphorylation at Ser768 is required for its inhibition of CFTR. In summary, our findings indicate that AMPK-dependent phosphorylation of CFTR inhibits CFTR activation by PKA, thereby tuning the PKA-responsiveness of CFTR to metabolic and other stresses in the cell.

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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

[hide] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. Wang G
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.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PMID:20952391]

Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

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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

[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. 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 [PMID:21059651]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.

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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

[hide] Structural models of CFTR-AMPK and CFTR-PKA intera... J Mol Model. 2011 Apr 1. Siwiak M, Edelman A, Zielenkiewicz P
Structural models of CFTR-AMPK and CFTR-PKA interactions: R-domain flexibility is a key factor in CFTR regulation.
J Mol Model. 2011 Apr 1., 2011-04-01 [PMID:21455600]

Abstract [show]
Cystic fibrosis (CF), the most common lethal genetic disease among Caucasians, is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR). CFTR's main role is to transport chloride ions across epithelial cell membranes. It also regulates many cell functions. However, the exact role of CFTR in cellular processes is not yet fully understood. It is recognized that a key factor in CFTR-related regulation is its phosphorylation state. The important kinases regulating CFTR are cAMP-dependent protein kinase A (PKA) and 5'-AMP-activated protein kinase (AMPK). PKA and AMPK have opposite effects on CFTR activity despite their highly similar structures and recognition motifs. Utilizing homology modeling, in silico mutagenesis and literature mining, we supplement available information regarding the atomic-resolution structures of PKA, AMPK and CFTR, and the complexes CFTR-PKA and CFTR-AMPK. The atomic-resolution structural predictions reveal an unexpected availability of CFTR Ser813 for phosphorylation by both PKA and AMPK. These results indicate the key role of the structural flexibility of the serine-rich R-domain in CFTR regulation by phosphorylation.

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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

[hide] Electrophysiological, biochemical, and bioinformat... Methods Mol Biol. 2011;741:443-69. Csanady L, Vergani P, Gulyas-Kovacs A, Gadsby DC
Electrophysiological, biochemical, and bioinformatic methods for studying CFTR channel gating and its regulation.
Methods Mol Biol. 2011;741:443-69., [PMID:21594801]

Abstract [show]
CFTR is the only member of the ABC (ATP-binding cassette) protein superfamily known to function as an ion channel. Most other ABC proteins are ATP-driven transporters, in which a cycle of ATP binding and hydrolysis, at intracellular nucleotide binding domains (NBDs), powers uphill substrate translocation across the membrane. In CFTR, this same ATP-driven cycle opens and closes a transmembrane pore through which chloride ions flow rapidly down their electrochemical gradient. Detailed analysis of the pattern of gating of CFTR channels thus offers the opportunity to learn about mechanisms of function not only of CFTR channels but also of their ABC transporter ancestors. In addition, CFTR channel gating is subject to complex regulation by kinase-mediated phosphorylation at multiple consensus sites in a cytoplasmic regulatory domain that is unique to CFTR. Here we offer a practical guide to extract useful information about the mechanisms that control opening and closing of CFTR channels: on how to plan (including information obtained from analysis of multiple sequence alignments), carry out, and analyze electrophysiological and biochemical experiments, as well as on how to circumvent potential pitfalls.

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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

[hide] CFTR regulation by phosphorylation. Methods Mol Biol. 2011;741:471-88. Alzamora R, King JD Jr, Hallows KR
CFTR regulation by phosphorylation.
Methods Mol Biol. 2011;741:471-88., [PMID:21594802]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is the gene product mutated in cystic fibrosis, a common lethal genetic disease characterized by abnormal electrolyte transport across epithelia. CFTR functions as an ATP-gated, phosphorylation-regulated Cl- channel that mediates agonist-stimulated apical membrane epithelial Cl- and bicarbonate secretion and also regulates a variety of other transport proteins and cellular processes. CFTR belongs to the ATP-binding cassette (ABC) transporter superfamily. Its presumed architecture consists of two transmembrane domain regions that form the channel pore, two nucleotide-binding domains that bind and hydrolyze ATP, and a unique regulatory (R) domain that contains numerous protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites. Other kinases have also been shown more recently to phosphorylate and regulate CFTR activity. This chapter describes strategies and methods for studying the phosphorylation of CFTR both in vitro and whole-cell systems.

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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

[hide] Control of CFTR channel gating by phosphorylation ... Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107. Gadsby DC, Nairn AC
Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis.
Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107., [PMID:9922377]

Abstract [show]
Control of CTFR Channel Gating by Phosphorylation and Nucleotide Hydrolysis. Physiol. Rev. 79, Suppl.: S77-S107, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is the protein product of the gene defective in cystic fibrosis, the most common lethal genetic disease among Caucasians. Unlike any other known ion channel, CFTR belongs to the ATP-binding cassette superfamily of transporters and, like all other family members, CFTR includes two cytoplasmic nucleotide-binding domains (NBDs), both of which bind and hydrolyze ATP. It appears that in a single open-close gating cycle, an individual CFTR channel hydrolyzes one ATP molecule at the NH2-terminal NBD to open the channel, and then binds and hydrolyzes a second ATP molecule at the COOH-terminal NBD to close the channel. This complex coordinated behavior of the two NBDs is orchestrated by multiple protein kinase A-dependent phosphorylation events, at least some of which occur within the third large cytoplasmic domain, called the regulatory domain. Two or more kinds of protein phosphatases selectively dephosphorylate distinct sites. Under appropriately controlled conditions of progressive phosphorylation or dephosphorylation, three functionally different phosphoforms of a single CFTR channel can be distinguished on the basis of channel opening and closing kinetics. Recording single CFTR channel currents affords an unprecedented opportunity to reproducibly examine, and manipulate, individual ATP hydrolysis cycles in a single molecule, in its natural environment, in real time.

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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

[hide] Role of individual R domain phosphorylation sites ... Biochim Biophys Acta. 2009 Jun;1788(6):1341-9. Epub 2009 Mar 26. Hegedus T, Aleksandrov A, Mengos A, Cui L, Jensen TJ, Riordan JR
Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A.
Biochim Biophys Acta. 2009 Jun;1788(6):1341-9. Epub 2009 Mar 26., [PMID:19328185]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.

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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

[hide] Computational studies reveal phosphorylation-depen... J Mol Biol. 2008 May 16;378(5):1052-63. Epub 2008 Mar 26. Hegedus T, Serohijos AW, Dokholyan NV, He L, Riordan JR
Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR.
J Mol Biol. 2008 May 16;378(5):1052-63. Epub 2008 Mar 26., [PMID:18423665]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its approximately 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.

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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

[hide] CFTR activation: additive effects of stimulatory a... Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33. Wilkinson DJ, Strong TV, Mansoura MK, Wood DL, Smith SS, Collins FS, Dawson DC
CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain.
Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33., [PMID:9252549]

Abstract [show]
To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, -712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased the half-maximal activation constant (KA) for activation by 3-isobutyl-1-methylxanthine, which is consistent with the hypothesis that phosphorylation at any of these sites promotes CFTR activation. The effect of substitution at serine-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Substitution at serine-641, -686, and -712 had no significant effect on activation sensitivity. The effects of multiple serine to alanine substitutions were consistent with the notion that phosphorylation at individual sites produced roughly additive effects, suggesting that the effect produced by phosphorylation of any one serine was not dependent on the phosphorylation state of other serines. These results are consistent with the notion that, although none of the phosphorylation sites studied here are absolutely necessary for activation of CFTR, individual sites contribute differently to the gating of the channel.

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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

[hide] Regulation of the cystic fibrosis transmembrane co... J Biol Chem. 1993 Sep 25;268(27):20259-67. Rich DP, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
J Biol Chem. 1993 Sep 25;268(27):20259-67., [PMID:7690753]

Abstract [show]
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

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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

[hide] Protein kinase A (PKA) still activates CFTR chlori... J Biol Chem. 1993 May 25;268(15):11304-11. Chang XB, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, Hanrahan JW, Riordan JR
Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.
J Biol Chem. 1993 May 25;268(15):11304-11., [PMID:7684377]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.

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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

[hide] Role of tyrosine phosphorylation in the muscarinic... J Biol Chem. 2013 Jul 26;288(30):21815-23. doi: 10.1074/jbc.M113.479360. Epub 2013 Jun 11. Billet A, Luo Y, Balghi H, Hanrahan JW
Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2013 Jul 26;288(30):21815-23. doi: 10.1074/jbc.M113.479360. Epub 2013 Jun 11., [PMID:23760269]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl(-) currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.

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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