ABCMdb

PMID: 15657296

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., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala Membrane conductance of resting and activated oocytes expressing WT or mutant S768A CFTR. Login to comment 146 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala (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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala (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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala The resulting time constants for WT (gray bars) and S768A (black bars) are summarized in Fig. 5 D. Login to comment 178 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala Activation in excised patches of macroscopic WT and S768A CFTR currents by low and high concentrations of PKA catalytic subunit. Login to comment 182 ABCC7 p.Ser768Ala (A and B) Currents recorded in patches containing hundreds of WT (A) or S768A (B) CFTR channels. Login to comment 185 ABCC7 p.Ser768Ala (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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala (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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala Kinetic behavior of WT and S768A CFTR channels in excised patches exposed to low and high [PKA]. Login to comment 199 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala (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 ABCC7 p.Ser768Ala against [PKA] for S768A and WT channels (Fig. 7). Login to comment 203 ABCC7 p.Ser768Ala Half-maximal activation of Po requires ‫051ف‬ nM PKA for WT channels, but only ‫07ف‬ nM PKA for S768A channels. Login to comment 204 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala Dependence on [PKA] of open probability, Po, of WT (᭹) and S768A (᭺) CFTR channels. Login to comment 214 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala The kinetics of R-domain phosphorylation, as demonstrated by the mobility shifts, was little altered in the two S768A mutants. Login to comment 221 ABCC7 p.Ser768Ala (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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala The increase in sensitivity was largest (approximately fivefold) for S768A mutant CFTR (Wilkinson et al., 1997). Login to comment 259 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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 ABCC7 p.Ser768Ala 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