Sealy Center for Structural Biology and Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-0437
Submitted 23 January 2004 ; accepted in final form 21 July 2004
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ABSTRACT |
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cystic fibrosis; chloride channel; protein kinases; ATP binding cassette proteins
The fact that R-domain phosphorylation is critical for the channel function of CFTR was documented by the following results: 1) CFTR is activated by PKA agonists in cells with native or heterologous expression (28, 3638, 41); 2) CFTR molecules lacking the R domain display Cl channel function after exposure to phosphorylated R domain but not when exposed to unphosphorylated R domain (27, 46); 3) there are eight conserved PKA phosphorylation consensus sites in the R domain, and five of them have been shown to be phosphorylated in vivo and in vitro by PKA (12, 32); 4) phosphorylation changes the secondary structure of the R domain in vitro (19); 5) serine-to-alanine substitutions in the PKA phosphorylation consensus sites of the R domain greatly attenuate CFTR activation in response to PKA stimulation (4).
In addition to its activation by PKA-mediated phosphorylation, CFTR could also be activated by other kinases such as PKC (9, 11, 15, 16, 23), PKG (6, 20), and calmodulin kinase (6, 32). However, the molecular mechanism of the activation of CFTR by kinases other than PKA and the interaction between PKA and non-PKA kinases in the activation of CFTR are not fully understood. These issues are important to understand from the points of view of the regulation of CFTR-mediated Cl transport by epithelial cells and the molecular mechanism of phosphorylation-mediated CFTR Cl channel gating. Recently, we found that Xenopus laevis (X)CFTR exhibits a severalfold higher response to PKC activation than to PKA activation when expressed in Xenopus oocytes (7). In the epithelial human (h)CFTR isoform, PKC stimulation potentiates the effect of PKA stimulation (40). In the present study, we confirmed the effect of PKC on the activation of XCFTR by PKA stimulation and then tried to ascertain the biophysical and molecular mechanisms of this effect. We found that PKC potentiates the activation of XCFTR expressed in Xenopus oocytes by PKA stimulation, i.e., the response to PKA stimulation subsequent to PKC stimulation was eightfold greater than that without previous PKC stimulation. This effect was not present in oocytes expressing hCFTR. To identify the biophysical mechanism, we assessed changes of channel number (N), single-channel conductance (), and single channel open probability (Po) of oocytes exposed to either PKA agonists or PKA and PKC agonists (potentiation). To identify the XCFTR regions responsible for the potentiation, we constructed hCFTR-XCFTR chimeras and tested them electrophysiologically. To our surprise, the XCFTR NBD2, and not the R domain, appears to be critical for the potentiation effect.
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MATERIALS AND METHODS |
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Dual-electrode voltage-clamp recordings.
Oocytes were placed in an oocyte-recording chamber (model RC-2Z; Warner Instruments, Hamden, CT). Bathing solutions were changed by a gravity-driven perfusion system at a rate of 1 ml/min. Oocytes were bathed in the HEPES-buffered solution ND96 (in mM: 96.0 NaCl, 2.0 KCl, 1.0 MgCl2, 1.8 CaCl2, and 5.0 HEPES-NaOH, pH 7.5). All experiments were performed at room temperature (2224°C). Borosilicate microelectrodes were pulled with a horizontal puller (model P-98; Sutter Instruments, Novato, CA), were filled with 2.0 M KCl, and had tip resistances of 0.51.5 M
when immersed in ND96 solution. A two-electrode voltage-clamp amplifier (model OC-725C; Warner Instruments) was used to amplify whole oocyte currents. Voltages were referenced to the bath. Oocytes were clamped at 40 mV. Membrane current and conductance were recorded using a setup described by Weber et al. (45). In short, two digital signal processing (DSP) boards, which were equipped with two high-speed (200 kHz) analog-to-digital converters and two digital-to-analog converters, were connected to the two-electrode voltage-clamp amplifier via a multifunctional interface controlled by the DSP boards. The membrane conductance (Gm) was calculated from the membrane current and voltage change in response to a 1.0-Hz, 5-mV sine wave.
Single-channel recording.
Single-channel recording protocols were adopted from Chan et al. (8) with some modifications. To remove the vitelline membrane, oocytes were shrunk for 10 min in stripping solution containing (in mM) 250 K-aspartate, 20 KCl, 1 MgCl2, 10 HEPES, and 1 EGTA, pH 7.4 with KOH. The vitelline membranes were removed manually with fine tweezers, and the oocytes were transferred to a recording chamber containing ND96 solution. Patch pipettes were pulled from borosilicate glass with a horizontal puller (same as above), Sylgard coated, and fire polished to a resistance of 610 M
(pipettes filled with pipette solution and immersed in ND96 solution). The pipette solution contained (in mM) 128 N-methyl-D-glucamine chloride (NMG-Cl), 2 MgCl2, 5 HEPES, and 0.1 GdCl2, titrated to pH 7.4 with HCl; 150- to 200-G
seals were obtained by gentle suction. The Ag-AgCl bath electrode was connected to the chamber bath by an agar bridge (2% agar in ND96). Outward unitary currents in CFTR channels were recorded at a pipette potential (Vp) of 40 mV, unless otherwise specified, via an Axopatch 200A amplifier (Axon Instruments, Union City, CA), filtered at 2 kHz with an eight-pole Bessel filter, and digitized online at 10 kHz with 100-Hz low pass by pCLAMP 8.2 (Axon Instruments).
The digitized, baseline-corrected currents from records containing one to five channels were idealized with the segmental K-means (SKM) algorithm (33) by Qub single-channel analysis software (State University of New York at Buffalo). Parameters of amplitude histograms and event occupancy were used to calculate and Po. Because of the outward rectification (5) in cell-attached patches,
values were estimated from amplitude histograms of cell-attached records at holding potentials of 20100 mV. In excised patches the currents recorded at holding potentials of 80 to +80 mV in symmetrical 140 mM Cl concentration were plotted against the voltage, and straight lines were fitted to yield conductances. Po values were calculated using the equation (14):
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Drugs.
To activate PKA, intracellular cAMP was elevated with a cAMP cocktail containing 250 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) and 25 µM forskolin (Sigma-Aldrich). To activate PKC, 250 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) was added to the bath. Stock solutions of these compounds were prepared in water (8-BrcAMP), dimethyl sulfoxide (PMA), or ethanol (forskolin) and diluted to the desired final concentration in ND96 solution immediately before use. [2-(Trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET+) was dissolved in bath solution to a 1 mM concentration immediately before use from a 1 M stock in water stored in 20°C. -Mercaptoethanol (
-ME) was dissolved in bath solution to a final 2 mM concentration immediately before use. At the concentrations used, the vehicles had no effects on the CFTR currents (not shown).
Statistical analysis. Data are expressed as means ± SE. Differences between means were compared by 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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In contrast with the above results, replacing the COOH-terminal region of XCFTR with the corresponding sequences from hCFTR in chimera E eliminated the PKC potentiation effect (Fig. 7), i.e., the conductance response to PKA agonists after PKC stimulation was the same as that before PKC stimulation. To narrow down the responsible region, we studied chimeras in which only the NBD2 (chimera F) or the COOH-terminal region distal to NBD2 (chimera G) was replaced with the corresponding sequence from hCFTR, respectively. In chimera F (Figs. 6B and 7) the potentiation disappeared, whereas in chimera G the potentiation (ratio of conductances with PKA activation after and before PKC stimulation = 4.7 ± 1.3; n = 4) persisted (Fig. 7). These results indicate that the NBD2 sequence is necessary for the kinase potentiation effect of XCFTR. To explore whether the COOH-terminal XCFTR sequence including NBD2 and the COOH-terminal region is sufficient to confer the PKC potentiation effect, we constructed a chimera (H) in which this XCFTR sequence replaced the corresponding hCFTR sequence. This chimera did not display the PKC potentiation effect (Fig. 7). These results indicate that NBD2 is necessary, but not sufficient, for the PKC potentiation effect observed in the XCFTR. Together, these results suggest that the molecular bases for the potentiation effect in XCFTR are complex (see DISCUSSION).
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DISCUSSION |
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The PKC potentiation in XCFTR is different from the PKC activation (7) and the PKC permissive effect (Ref. 23 and below). Our previous results (7) demonstrated that PKC activation in XCFTR depends on a single PKC phosphorylation consensus site (T665LRR) in the R domain. A PKC consensus site at this location exists in CFTR from all species so far sequenced (from dogfish to chimpanzee) except human and zebra fish (TLHR). We found that the activation by PKC stimulation was abolished in the mutant T665A XCFTR and transferred to the hCFTR mutant TLHR TLRR (7). The chimera in which the XCFTR R domain was replaced with the corresponding hCFTR sequence (chimera B) lacks the PKC activation site; nevertheless, it exhibits potentiation, indicating that these two processes have different molecular bases. A third phenomenon is the requirement for a certain basal level of PKC activity for the activation of CFTR by PKA stimulation, named the PKC permissive effect by Hanrahans group (23). This effect, present in both XCFTR and hCFTR, depends on several PKC phosphorylation consensus sites in the R domain and NBD1 (10, 13). Hence, the three modes by which PKC stimulation affects CFTR function can be distinguished because of the ortholog specificity (hCFTR vs. XCFTR) or because of the different amino acid residues involved.
Concerning the biophysical mechanism of potentiation, we tested whether the insertion of new channels plays a role. Channel insertion has been assessed from changes in plasma membrane electrical capacitance in parallel with increases in conductance, which are interpreted to result from vesicle fusion (26, 42, 45). A problem with this method is that accurate measurements of membrane capacitance are difficult, in particular when Gm is changing, as is the case with the stimulation of CFTR by kinase agonists (12). In addition, the possibility exists that the membrane vesicles fusing with the membrane do not contain the channels. In our hands, there were no consistent correlations between the apparent capacitance and conductance changes during kinase stimulation (data not shown). Membrane protein labeling and detection by immunofluorescence or Western blot analysis has also been used to address this question. A problem with this approach is that the labeling process is slow, taking tens of minutes to hours, and therefore makes it difficult to assess acute effects of kinase stimulation. In addition, this methodology does not allow for identification of functional copies of the protein under study. For these reasons, we used the substituted-cysteine-accessibility method (1, 2, 24) to assess whether or not the potentiation phenomenon involves CFTR channel insertion. The results with the substituted-cysteine-accessibility method indicate that insertion of new channels does not play a significant role in the potentiation effect. These results extend the observations of Dawsons group (24), who showed a lack of insertion of new channels when hCFTR-expressing oocytes were treated with PKA agonists. Confirming their observations, we also obtained a negative result with PKA stimulation (data not shown). We conclude that the proposal of channel insertion based on increases in membrane capacitance observed when oocytes expressing hCFTR are exposed to PKA agonists (24, 42, 45) is incorrect. Either the assessment of the capacitance is in error (12) or vesicles fuse with the plasma membrane but do not contain activatable CFTR molecules. An important point is that exocytic insertion of CFTR channels may vary among cell types and/or among orthologs (25). Thus the results of Dawsons group and ours, obtained in Xenopus oocytes, cannot be generalized to other systems.
Both single-channel and Po are increased in response to PKA stimulation after PKC stimulation. The change in Po was quite large, indicating that this is the main mechanism of increase of macroscopic conductance; the change in
was small, but significant, and we also observed it after stimulation of XCFTR with PKC agonist alone (13). There was a significant difference between the relative changes in whole cell conductance and single-channel properties. However, these numbers may not be directly comparable because of the faster superfusion of the oocytes in the whole cell experiments and difficulties in determining the time of maximal stimulation of on-cell CFTR channels. Regulation of ion channels by PKC-mediated phosphorylation is not generally expected to involve changes in single-channel
, but it has been observed in a few instances (18, 29, 30).
The R-domain sequence differences between XCFTR and hCFTR are not responsible for the lack of kinase potentiation effect in hCFTR. This is a surprising result, given the dominant role of the R domain in the regulation of CFTR by phosphorylation (9, 31, 32). Interestingly, Chang et al. (9) noted that hCFTR could still be activated by PKA stimulation in a mutant in which all PKA consensus phosphorylation sites were knocked out in the R domain. Also, both hCFTR and XCFTR could be partially activated by PKA or PKC stimulation when Ser/Thr residues in all PKC phosphorylation consensus sites in the R domain were mutated to Ala (13). Possible explanations for these results are phosphorylation of cryptic sites outside the R domain, indirect phosphorylation of CFTR by another kinase activated by PKA or PKC, or a protein kinase-sensitive accessory protein that would interact with CFTR. All these possibilities could in principle apply to the kinase potentiation effect on XCFTR.
Even if the effect of kinase stimulation on CFTR requires phosphorylation of one or more unknown accessory proteins, the accessory protein(s) must interact with CFTR molecules to change CFTR channel function. Thus the different PKC potentiation effects in hCFTR and XCFTR must stem from the difference in the structure of these two CFTR homologs. Elimination of the PKC potentiation effect of XCFTR in chimeras in which NBD2 was replaced by the corresponding hCFTR sequences indicates that the difference between the NBD2s of XCFTR and hCFTR is critical for the PKC potentiation effect observed in XCFTR. The fact that replacement of the hCFTR sequence beyond the beginning of NBD2 with the corresponding XCFTR sequence did not confer the PKC potentiation effect indicates that the NBD2 difference is not sufficient for this effect. It is possible that other domains are involved; these domains would have a positive effect in XCFTR and/or a negative effect in hCFTR, always in association with XCFTR NBD2. The significant decrease of the potentiation effect in chimeras A and C (compared with XCFTR) suggests that the R domain and the NH2-terminal domain may also be involved. In contrast, the differences of the NBD1s, TM5 and 6, and the COOH-terminal region of XCFTR are not necessary for the PKC potentiation effect. Additional experiments will be required to identify the specific sequence in NBD2 that is responsible for the PKC potentiation effect and the roles of other CFTR domains.
The molecular basis for the critical role of NBD2 in mediating the PKC potentiation effect remains unknown. One possibility is a difference in NBD2 phosphorylation between XCFTR and hCFTR. There are five PKA or PKC phosphorylation consensus sites in XCFTR NBD2 (2 of them are also present in hCFTR), but there is currently no evidence for phosphorylation of these sites. In contrast, the critical sites for ATP binding and hydrolysis in NBD2 are conserved in these two molecules. Another possibility is phosphorylation of accessory protein(s) that may interact with NBD2. Already-known CFTR accessory proteins such as CAP70 and syntaxin 1A are unlikely candidates because they do not interact with the NBD2 of hCFTR (22, 44).
In summary, we found that the response to PKA stimulation increases severalfold by preexposure of XCFTR to PKC agonist. The biophysical bases for the potentiated macroscopic response to PKA stimulation are a large increase in single-channel Po and a modest increase in single-channel with no detectable change in number of channels in the plasma membrane. The differences between the NBD2s of hCFTR and XCFTR are critical for the XCFTR-specific PKC potentiation effect, and the NH2-terminal domain and R domain are not essential but may participate in the effect. Future efforts to dissect the molecular mechanism of the potentiation effect may help our understanding of the regulation of CFTR by phosphorylation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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