ABCMdb

PMID: 9305845

Winter MC, Welsh MJ
Stimulation of CFTR activity by its phosphorylated R domain.
Nature. 1997 Sep 18;389(6648):294-6., [PubMed]

Sentences

No. Mutations Sentence Comment

124 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala Here we test this by adding an R-domain peptide to a CFTR variant in which much of the R domain had been deleted (CFTR-DR/S660A): in contrast to predictions, we found that adding an unphosphorylated R domain to CFTR-DR/S660A did not inhibit activity, whereas a phosphorylated R-domain peptide stimulated activity. Login to comment 125 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity. We previously studied a CFTR variant, CFTR-DR/S660A, in which much of the R domain (residues 708-835) was deleted and the phosphorylation site was removed at Ser 660 by mutation to alanine4 . Login to comment 126 ABCC7 p.Ser660Ala To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity. We previously studied a CFTR variant, CFTR-DR/S660A, in which much of the R domain (residues 708-835) was deleted and the phosphorylation site was removed at Ser 660 by mutation to alanine4 . Login to comment 128 ABCC7 p.Ser660Ala This model predicts that the activity of CFTR-DR/S660A should be similar to that of phosphorylated wild-type CFTR. Login to comment 129 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala However, when we examined the single-channel properties of CFTR-DR/ S660A, we found that Po was 0:13 Ϯ 0:01 (1 mM ATP, n ¼ 7), much lower than that of phosphorylated wild-type CFTR (Po ¼ 0:46 Ϯ 0:02; 1 mM ATP, n ¼ 17, P Ͻ 0:001). Login to comment 130 ABCC7 p.Ser660Ala However, when we examined the single-channel properties of CFTR-DR/ S660A, we found that Po was 0:13 6 0:01 (1 mM ATP, n &#bc; 7), much lower than that of phosphorylated wild-type CFTR (Po &#bc; 0:46 6 0:02; 1 mM ATP, n &#bc; 17, P , 0:001). Login to comment 131 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala But deletion of the R domain in CFTR-DR/S660A could still have additional nonspecific effects on channel structure which might reduce activity. We considered, therefore, that the phosphorylated R domain might stimulate the channel and that the defective functioning of CFTR-DR/S660A is due to its lacking a phosphorylated R domain. Login to comment 132 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala To test this idea, we added a recombinant R domain (R1, residues 645-834, similar to a peptide reported previously10 ) to the cytosolic surface of CFTR-DR/S660A and measured activity by using the excised, inside-out patch-clamp technique. Login to comment 133 ABCC7 p.Ser660Ala To test this idea, we added a recombinant R domain (R1, residues 645-834, similar to a peptide reported previously10 ) to the cytosolic surface of CFTR-DR/S660A and measured activity by using the excised, inside-out patch-clamp technique. Login to comment 136 ABCC7 p.Ser660Ala Additional evidence that the effect of phosphorylation was on R1 rather than on CFTR-DR/S660A came from our finding that addition of phosphorylated R1 in the presence of protein-kinase-inhibitor peptide under conditions that block the effect of PKA11 gave similar results (43:2 Ϯ 3:4% increase; n ¼ 3). Login to comment 137 ABCC7 p.Ser660Ala Additional evidence that the effect of phosphorylation was on R1 rather than on CFTR-DR/S660A came from our finding that addition of phosphorylated R1 in the presence of protein-kinase-inhibitor peptide under conditions that block the effect of PKA11 gave similar results (43:2 6 3:4% increase; n &#bc; 3). Login to comment 141 ABCC7 p.Ser660Ala Our results also suggest that the low Po found for CFTR-DR/S660A results at least in part from a lack of stimulation by a phosphorylated R domain, rather than from nonspecific structural changes. Login to comment 142 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala The fact that R1 did not increase the Po of CFTR-DR/S660A to wild-type CFTR values may be because the endogenous R domain is constrained in the correct orientation by the site of interaction; the interaction of exogenously applied R1 with CFTR-DR/S660A is probably less efficient. Login to comment 143 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala Our results contrast with a report that addition of an in vitro translation mixture containing a different unphosphorylated R domain (residues letters to nature 294 NATURE |VOL 389 |18 SEPTEMBER 1997 a -20 -10 0 I(pA) 5 min R1 PKA b-30 -20 -10 0 5 min R1 ∆R/S660A Wild type 0 10 20 30 40 50 - + - + c * PKA I(pA)ChangewithR1addition(%) Figure 1 Effect of R1 on CFTR-DR/S660A and wild-type CFTR. Login to comment 144 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala a, b, Time course of Cl- current in excised, inside-out patches containing many CFTR-DR/S660A channels during addition of PKA and R1 (50 nM), as indicated by bars. Login to comment 148 ABCC7 p.Ser660Ala c, Effect of R1 in the presence and absence of PKA on current measured from CFTR-DR/S660A and wild-type CFTR. Login to comment 152 ABCC7 p.Ser660Ala For CFTR-DR/S660A, n is 10 and 9; for wild-type CFTR, n is 4 and 5, in the absence or presence of PKA, respectively. Login to comment 156 ABCC7 p.Ser660Ala To find out how the phosphorylated R domain stimulates CFTR-DR/S660A, we investigated channel gating. Login to comment 158 ABCC7 p.Ser660Ala Figure 2 shows that phosphorylated R1 increases the rate at which CFTR-DR/S660A opened into bursts, without affecting the rate of closure from bursts. Login to comment 162 ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala Mutation of the individual serines reduced Po, but to different extents: the S795A mutation had the largest effect and S737A had little or no effect in the presence of 1 mM ATP (Fig. 3a, b). Login to comment 166 ABCC7 p.Ser660Ala Thus addition of the phosphorylated R domain to CFTR-DR/ S660A and mutation of phosphorylation sites in wild-type CFTR had opposite effects on the same kinetic step in channel gating, the rate of channel opening. Login to comment 170 ABCC7 p.Ser660Ala Figure 4 shows that the mutations reduced Po at low concentrations of ATP; Po was letters to nature NATURE |VOL 389 |18 SEPTEMBER 1997 295 Figure 2 Effect of R1 on CFTR-DR/S660A channels in the presence of PKA. Login to comment 172 ABCC7 p.Ser660Ala b, c, Effect of R1 on the rates of entry into and exit from a burst for CFTR-DR/S660A channels in the presence of PKA. Login to comment 179 ABCC7 p.Ser795Ala a, Example of continuous single-channel tracings from wild-type CFTR and CRTR- S795A; dashed lines indicate the closed state. Login to comment 185 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala Number of experiments for b/c and d were: 17/6 for wild-type (WT), 8 for wild-type (-PKA), 8/7 for S660A, 5/5 for S737A, 8/6 for S795A, 9/7 for S813A, and 11/4 for S-Quad-A. Login to comment 188 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Po 0 0.2 0.4 0.6 0.8 1 10 [ATP] (mM) S813A S795A S737A S660A WT (-PKA) WT Figure 4 Effect of ATP concentration on Po of CFTR containing phosphorylation site mutations. Login to comment 193 ABCC7 p.Ser660Ala This might also be true for CFTR-DR/S660A. Login to comment 200 ABCC7 p.Ser660Ala When phosphorylation modifies the R domain, it might have two effects: the first could be permissive, releasing a steric inhibition, consistent with our finding that deletion of the R domain produces a channel that is partially active; the second effect might be stimulatory, facilitating interaction of the NBDs with ATP, which would be consistent with results we obtained when a phosphorylated R domain was added to CFTR-DR/S660A and with the effects of CFTR-containing mutations in the endogenous R domain. Login to comment