Correspondence to: Guillermo A. Altenberg, Department of Physiology and Biophysics, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0437. Fax:(409) 772-1301 E-mail:galtenbe{at}utmb.edu.
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Abstract |
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Mutations of the CFTR, a phosphorylation-regulated Cl- channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.
Key Words: chloride channel, PKA, ABC proteins, R domain
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INTRODUCTION |
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Cystic fibrosis results from mutations in the CFTR, a 1,480amino acid protein that functions as a Cl--selective ion channel in a variety of epithelial cells. CFTR has two topologically equivalent halves, each consisting of a transmembrane domain (six membrane-spanning helices) followed by a nucleotide-binding domain. The cytoplasmic regulatory domain (R domain)1 links these two halves. The membrane-spanning helices form the anion-conductive pore, the nucleotide-binding domains bind and hydrolyze ATP and are involved in channel gating, and the R domain contains consensus phosphorylation sites for PKA and PKC (for review see
CFTR channels are gated by exposure to PKA and ATP, both in excised patches and after purification and reconstitution in planar lipid bilayers (for review see
PKA-mediated activation of human CFTR (hCFTR) requires endogenous phosphorylation by PKC (
The goal of the present work was to determine the molecular bases for the differences in the activation of hCFTR and XCFTR by PKC stimulation. Our results show that, in contrast with the complexity of PKA-mediated activation, the stimulation of XCFTR by PKC depends on a single PKC consensus phosphorylation site in the R domain. Confirming the importance of the critical site in XCFTR, engineering an equivalent site in hCFTR makes this molecule responsive to PKC stimulation.
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MATERIALS AND METHODS |
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cRNA Preparation
Full-length hCFTR and XCFTR cDNAs were subcloned into the oocyte expression vector pOCYT7 (gift from Dr. Nancy Wills, University of Texas Medical Branch;
Oocyte Preparation and cRNA Injection
Xenopus laevis oocytes were isolated and prepared using a well-documented protocol (
Two-electrode Voltage Clamp
Oocytes were bathed in the HEPES-buffered solution ND96 ([in mM] 96 NaCl, 2 KCl, 1.0 MgCl2, 1.8 CaCl2, and 5 HEPES/NaOH, pH 7.40). All experiments were performed at 2224°C. Borosilicate microelectrodes were pulled with a horizontal puller (P-97; Sutter Instruments), filled with 3 M KCl, and had tip resistances of 0.51.5 M when immersed in ND96 solution. A voltage-clamp amplifier (model OC-725C; Warner Instruments) was used to measure whole-oocyte conductance. Voltages were referenced to the bath. Membrane currents were filtered at 1.0 kHz, digitized, stored, and analyzed with pCLAMP version 8.0 (Axon Instruments). I-V relationships were obtained by current measurements 400 ms after changing the potential from a holding value of -30 mV to test values ranging from -100 to +30 mV, in 10-mV steps, with 100-ms intervals between pulses. Oocyte conductance was determined in the consistently linear range between the reversal potential and 30 mV. The membrane conductances of unstimulated oocytes (i.e., no 8-Br-cAMP or PMA) after cRNA injection were not different from those of water-injected oocytes (Table 1). In hCFTR- and XCFTR-expressing oocytes, membrane conductances were directly proportional to the amount of cRNA injected (not shown) and time after injection (Table 1). In all experiments detailed here, supra-maximal concentrations of either cAMP cocktail (250 µM 8-Br-cAMP and 25 µM forskolin) or PMA (250 nM) were used to stimulate maximally PKA or PKC, respectively. For the time course experiments, differences between the current at -30 mV (the holding potential) and 0 mV were measured every 5 s. Halide selectivity sequences were determined from the changes in reversal potential after substituting NaCl with the corresponding sodium-halide salt for 2 min before the I-V plots. Corrections for liquid junction potentials were as previously described (
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Whole-cell Patch Clamp
Whole cell patch-clamp studies were performed essentially as previously described ( tip resistance in nearly symmetric NMDG-Cl) were pulled with a horizontal pipet puller (model P-97; Sutter Instruments). Negative pressure was used to rupture the membrane patch after obtaining a gigaohm seal, and currents were measured in the whole-cell configuration with an amplifier (model Axopatch 200A; Axon Instruments). The holding voltage (Vm) was -60 mV. I-V relationships were constructed using 400-ms voltage pulses between -80 to +80 mV, at 10-mV intervals. PClamp6 (Axon Instruments) was used for generation of the pulses, data collection, and analysis.
Human-Xenopus R Domain Chimera Construction
A chimera, in which residues 607811 of the hCFTR were replaced by the corresponding XCFTR residues, was engineered by a combination of site-directed mutagenesis and PCR. Site-directed mutagenesis was used to introduce MluI and KasI unique sites at the 5' and 3' ends of the R domain hCFTR cDNA sequence with silent mutations. The QuickChange site-directed mutagenesis kit (Stratagene) was used to insert the unique restriction sites. Two mutagenic oligonucleotide primers, each complementary to opposite strands of the vector, were used for each mutagenesis reaction. The wild-type hCFTR was used as a template. The primers used were as follows (only sense primer is shown, restriction sites underlined): 5'-TGGCTAACAAAACGCGTATTTTGGTCAC-3' (MluI site) and 5'-GGATATATATTCAAGGCGCCTATCTCAAGAAAC-3' (KasI site). The reaction mixture was heated at 95°C for 45 s and subjected to 12 cycles for 30 s at 95°C, 30 s at 55°C, and 10 min at 72°C. Cycling was finished by a 10-min incubation at 70°C. The PCR reaction was treated with DpnI to digest the DNA template, and then used to transform Escherichia coli. The XCFTR R domain cDNA was amplified by PCR, introducing MluI and KasI sites that were used to exchange the hCFTR and XCFTR R domain sequences. The PCR primers were as follows (MluI and KasI sites underlined): 5'-GGGACGCGTATTTTAGTTACATCT AAAGTCG-3' (forward) and 5'-TTTGAAGTGGATATATATAATAGGCGCCGCG-3' (reverse).
PKC Consensus Site Mutagenesis
The QuickChange site-directed mutagenesis kit was used, as described above, to make point mutations. To remove consensus phosphorylation sites, Ser or Thr were substituted with Ala. The primers also contained silent mutations to introduce unique restriction sites for primary screening. The following primers were used (only sense primer is shown, with mutations underlined): 5'-TAATAACTGAGGCCCTGAGACGATGCT-3' (Thr665 to Ala, plus addition of HaeIII site), 5'-GTCAAGAATAAAGCTTTTAAGCAGG-3' (Ser686 to Ala, plus addition of HindIII site), 5'-TGGGGATTTCGCTGAGAAAAGAAAGAG-3' (Ser694 to Ala, plus addition of DdeI site), and 5'-CAAGAAAAACTGCAGTTCGTAAAATG-3' (Ser790 to Ala, plus addition of PstI site). For construction of the H667R-hCFTR mutant, the primer used was 5'-AGACCTTGCGCCGTTTCTCA-3' (construct screened with HhaI, underlined). The wild-type XCFTR or hCFTR in the pOCYT7 vector (see above) was used as a template.
Oocyte cAMP Levels
Intracellular cAMP was determined 2448 h after cRNA injection. Oocytes were incubated for 20 min in the presence of either 250 nM PMA to activate PKC or 25 µM forskolin to activate PKA. At the end of the incubation period, cells were lysed by sonication. All samples were heated to 95°C for 5 min and centrifuged at 12,000 g for 15 min at 4°C. The supernatants were assayed using a cAMP enzyme immunoassay kit (Amersham Pharmacia Biotech).
Western Blot Analysis
Western blot analysis was performed with the affinity-purified rabbit anti-CFTR antibody 1468 (provided by Dr. Jonathan Cohn, Duke University, Durham, NC;
Solutions and Drugs
To activate PKA, intracellular cAMP was elevated using a cAMP cocktail containing 250 µM 8-Br-cAMP and 25 µM forskolin (Sigma-Aldrich). To activate PKC, 250 nM PMA (Sigma-Aldrich) was added to the bath. Stock solutions of these compounds were prepared in water (8-Br-cAMP), dimethylsulfoxide (PMA), or ethanol (forskolin), and diluted to the desired final concentration in ND96 solution immediately before use. At the concentrations used, the vehicles had no effects on the CFTR currents.
Statistical Analysis
Data are expressed as the means ± SEM. Differences between means were compared by either paired or unpaired two-tailed t tests, as appropriate. Statistical significance was ascribed to P < 0.05.
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RESULTS |
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Differential Activation of Human and Xenopus CFTR by PKC
We expressed human and Xenopus CFTR orthologues in Xenopus oocytes because these cells are capable of appropriate posttranslational processing, express CFTR at high levels, and have no significant endogenous cAMP- or PMA-activated Cl- currents ( Cl- > I- > F- for hCFTR and I- > Cl- > Br- > F- for XCFTR [ Fig 1C and Fig D]), and their reversal potentials (in NaCl-based bath solution) near the Cl- equilibrium potential (about -30 mV;
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To determine the effect of PKC-mediated phosphorylation on the activation of the two CFTR orthologues, oocytes expressing hCFTR or XCFTR were exposed to PMA. I-V relationships, current time course, and conductance data in Fig 2 show that, in hCFTR-expressing oocytes, PMA alone elicits only a small fraction of the conductance observed after the subsequent application of the cAMP cocktail. Exposure to PMA resulted, as previously observed by others (
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In contrast, in XCFTR-expressing oocytes, PMA activated 79 ± 8% of the conductance seen after stimulation of both PKA and PKC (Fig 2B, Fig D, and Fig E). The PMA-activated conductance was sustained for over 30 min, in the continued presence of PMA, and was reversible after extended washout, like the XCFTR conductance elicited by PKA stimulation (not shown). In these experiments, higher 8-Br-cAMP concentrations did not increase the currents further. Relative to the current elicited by 250 µM 8-Br-cAMP, the current after 500 µM 8-Br-cAMP was 98 ± 12% (n = 3), and after 1 mM 8-Br-cAMP it was 96 ± 2% (n = 3).
The above results demonstrate that exposure to PMA activates XCFTR to a level similar to that obtained after PKA stimulation. To determine whether the effect of PMA is due to activation of PKC, we tested the inactive phorbol analogue 4-PMA on XCFTR-expressing oocytes. Application of 4
-PMA (250 nM) did not change oocyte membrane conductance, whereas subsequent exposure to 4ß-PMA caused an increase in conductance to 86 ± 11% of the value after exposure to the cAMP cocktail and 4ß-PMA combined, similar to the results in Fig 2. Further, application of 4ß-PMA to water- or hCFTR-injected oocytes, as well as application of 4
-PMA after 4ß-PMA in the XCFTR-injected oocytes, had no effect on the oocyte conductances (data not shown).
Another possibility is that PMA increases XCFTR conductance via an elevation of cAMP. Cross-talk between the PKA and PKC signaling systems has been demonstrated in a number of systems (
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Finally, the difference between hCFTR and XCFTR sensitivity to PMA could be in principle explained by the expression system used. One or more cell-specific components in Xenopus oocytes could allow XCFTR, but not hCFTR, to be directly or indirectly sensitive to PKC-mediated phosphorylation. This question was addressed by experiments in which either XCFTR or hCFTR was expressed in COS-1 cells. In four experiments in cells transfected with XCFTR cDNA, PMA elicited a conductance of 55 ± 3% of that produced by 8-Br-cAMP or forskolin.2 In contrast, in COS-1 cells expressing hCFTR, the increase in conductance elicited by PMA was on average 21 ± 4%. Taken together, the experiments in Xenopus oocytes and COS-1 cells demonstrate that the difference in PKC-mediated activation of human and Xenopus orthologues is inherent to the CFTR molecules and not to the host cells.
The R Domain Accounts for the Differences in the Responses of Human and Xenopus CFTR to PKC Activation
To test for a possible role of the R domain, a chimera was constructed in which residues 607 811 of hCFTR (Fig 4 and Fig 5 A) were replaced with the equivalent residues of XCFTR. This chimera (HXH-CFTR) contains the NH2- and COOH-terminal transmembrane domains and nucleotide-binding domains from hCFTR. It should exhibit the halide-selectivity sequence of hCFTR, because previous experiments with human/Xenopus chimeras revealed that the halide selectivity sequence of hCFTR and XCFTR depends on the NH2-terminal transmembrane domain (
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HXH-CFTR was fully processed and inserted in the plasma membranes of Xenopus oocytes as shown by Western blot analysis of plasma membranes (Fig 5 B). Electrophysiological studies showed no appreciable current in the absence of kinase stimulation (Fig 5 C and 6 A), as shown above for wild-type hCFTR and XCFTR (Fig 1 and Fig 2). The PKA agonist 8-Br-cAMP reversibly elicited a large, near-linear current, with a reversal potential of approximately -30 mV (Fig 6A and Fig C) and halide-selectivity sequence identical to that of wild-type hCFTR (Fig 5 D).
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The response to PMA of oocytes expressing HXH-CFTR was similar to those of oocytes expressing either hCFTR or XCFTR, in a clearly bimodal distribution, without intermediate responses. A basal level of PKA-mediated phosphorylation, insufficient to activate XCFTR by itself, is required for the PMA effect (
Conserved Phosphorylation Sites Are Not Involved in the Response to PKC Activation
As shown in Fig 4, the R domain of XCFTR contains at least seven consensus sites for phosphorylation by PKC. Four of the phosphorylatable residues are also present in hCFTR (Thr605, Ser686, Ser707, and Ser790), and two of them (Ser686 and Ser790) are phosphorylated in vitro in hCFTR (
Substitution of the two conserved serine residues known to be phosphorylated (Ser686 and Ser790;
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Thr665 Is Essential for the Response to PKC Activation of XCFTR
To investigate the roles of the unique PKC consensus sites (Thr665 and Ser694), we expressed the mutant lacking both sites (T665A/S694A-XCFTR) in Xenopus oocytes. This mutant was also well expressed, did not exhibit basal current, and was sensitive to agonists of PKA-mediated phosphorylation (Fig 7 B). However, PMA elicited a small current (Fig 7B and Fig C) that was not different from that measured in oocytes expressing wild-type hCFTR. The lack of response to PMA could not be overcome by a higher concentration (750 nM, data not shown). This result suggests that at least one of the two unique sites in the R domain of XCFTR is critical for activation by PKC-mediated phosphorylation. Although the degrees of activation of this mutant by PKA and PKC were the same as in the wild-type hCFTR, its halide-selectivity sequence (I- > Cl- > Br- > F-; Fig 1) was identical to that of the wild-type XCFTR, as expected.
To ascertain the roles of the two conserved sites, we mutated Thr665 and Ser694 separately and tested the effect of PMA in primed oocytes. When S694A-XCFTR was stimulated with PMA, there was full current activation (Fig 8A and Fig C). In contrast, in oocytes expressing T665A-XCFTR, PMA failed to produce full current activation (Fig 8B and Fig C). Currents in cRNA-injected oocytes vary from cell to cell. Thus, it is possible that the reduced activation by PMA of XCFTR mutants containing the Thr665 to Ala mutation is not absolute, but relative to the activation by PKA stimulation (i.e., the mutation increases the activation of CFTR by cAMP). The data in Fig 9 indicate that this is not the case because the cAMP-activated currents are not different among oocytes expressing wild-type XCFTR, S686A/S790A-XCFTR, or T665A/S694A-XCFTR. These results prove that Thr665 is the residue necessary for the activation of CFTR by PKC.
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Engineering a PKC Consensus Phosphorylation Site that Includes Thr665 Confers PKC-sensitivity to hCFTR
The hCFTR is unique among all known CFTR homologues in that it lacks a PKC consensus site at the location equivalent to residue 665 in XCFTR. As shown in Fig 4, this is due to the presence of His667 instead of arginine or lysine. Since Thr665 is present in hCFTR, we decided to determine whether the formation of a PKC consensus phosphorylation site in hCFTR by substituting His with Arg at position 667 is sufficient to confer the Xenopus phenotype to hCFTR. The mutant H667R-hCFTR was expressed in Xenopus oocytes and tested for activation by PKC stimulation. In oocytes primed with 8-Br-cAMP, as described above, PMA produced a large activation of the conductance, amounting to 68 ± 6% (n = 6) of the current elicited by the combination of PMA and cAMP cocktail (Fig 10). The PMA-dependent conductance in the presence of priming was significantly greater than that in wild-type hCFTR, although perhaps smaller than the analogous value in HXH-CFTR. This demonstrates that the phosphorylation site at Thr665 is sufficient to explain CFTR activation by PKC-mediated phosphorylation.
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DISCUSSION |
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The phosphorylation of serines and threonines by cAMP-dependent kinases is a conserved step in the activation of CFTR Cl- channels from several species (
PKC stimulation alone produced only a small activation of hCFTR expressed in Xenopus oocytes, a result consistent with those previously reported using either heterologous expression systems or native CFTR-expressing cells (
A Single PKC Consensus Phosphorylation Site in the R Domain Is Needed for the Activation of Xenopus CFTR by PKC Stimulation
The chimera in which the R domain of XCFTR replaced the R domain of hCFTR exhibited the halide-selectivity sequence of hCFTR, as expected from previous studies indicating that the ion selectivity of CFTR is determined by the NH2-terminal transmembrane spanning domain (
The mutagenesis experiments presented in this paper indicate that the PKC consensus phosphorylation site including Thr665 is required for the activation of the channel by PKC stimulation. In hCFTR, Thr665 is conserved, but the PKC consensus phosphorylation site is missing. Insertion of this site in the H667R-hCFTR mutant resulted in a large increase in current in response to PKC activation. The experiments on this mutant and the HXH chimera were different in some respects (e.g., priming concentration of 8-Br-cAMP and amount of cRNA injected), making a quantitative comparison of the responses to PKC activation difficult. In any event, the results indicate that the PKC consensus phosphorylation site that includes Thr665 is critical for the response to PMA. If the activation of the H667R-hCFTR mutant is in fact less than that of the HXH chimera, then one would conclude also that the phosphorylation of Thr665, or its functional effect, involves other residues of the R domain of CFTR.
PKC-mediated activation of hCFTR requires endogenous phosphorylation by PKA (
Our data suggest, but do not prove, that the stimulation of XCFTR by PMA is directly mediated by phosphorylation of Thr665 by PKC. This is the simplest explanation of our results because of the following reasons. First, the inactive phorbol ester 4-PMA is ineffective, ruling out most nonspecific effects of PMA. Second, PMA treatment does not increase cAMP levels in oocytes, ruling out an indirect effect via PKA activation. Third, cGMP is ineffective in Necturus gallbladder epithelial cells (
The Mechanism of CFTR Activation by Phosphorylation of the R Domain
It is clear that hCFTR is activated by PKA-mediated phosphorylation of residues located mainly in the R domain (
It has been assumed that exon 13 of hCFTR (residues 591830) codes for the R domain (
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Footnotes |
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1 Abbreviations used in this paper: hCFTR, human CFTR; HXH-CFTR, CFTR chimera containing the transmembrane domains and nucleotide-binding domains of hCFTR and the regulatory domain of Xenopus CFTR; R domain, regulatory domain; XCFTR, Xenopus CFTR.
2 In one experiment, there was no response to PMA. If this experiment is included in the calculation, the PMA-stimulated conductance is still significantly increased, i.e., 47 ± 4% of the 8-Br-cAMP-stimulated current. A negative result was also observed in 1 of 16 experiments in Xenopus oocytes expressing XCFTR. A possible explanation for the occasional absence of response to PMA is that the cells had low endogenous cAMP levels (see next page).
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Acknowledgements |
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We thank Drs. B.N. Christensen, S.A. Lewis, and S.A. Weinman for comments on preliminary versions of this manuscript, Dr. G. Zhang and Mr. S. Govani for help with the experimental work, and Ms. L. Durant for secretarial help.
This work was supported by National Institutes of Health grants No. DK-38734 (to L. Reuss) and No. CA-72783 (to G. Altenberg). B. Button is a Jeane Kempner Fellow.
Submitted: 17 January 2001
Revised: 30 March 2001
Accepted: 4 April 2001
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