Sealy Center for Structural Biology and Department of Neuroscience and Cell Biology, The University of Texas Medical Branch, Galveston, Texas 77555-0437
Submitted 10 May 2004 ; accepted in final form 25 June 2004
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ABSTRACT |
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chloride channel; channel regulation; cystic fibrosis transmembrane conductance regulator gating; cystic fibrosis; phosphorylation; protein kinase A
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MATERIALS AND METHODS |
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Oocyte preparation and cRNA injection. The protocol for preparing oocytes was previously described (6). In brief, Xenopus laevis ovarian lobes were surgically removed from anesthetized frogs and treated with 0.7 mg/ml type IV collagenase (Sigma-Aldrich, St. Louis, MO) in Barth's solution (in mM: 88 NaCl, 1.0 KCl, 1.0 CaCl2, 1.0 MgCl2, and 10 HEPES-NaOH, pH 7.4) containing (in mg/ml) 10 penicillin, 10 streptomycin, and 100 gentamicin sulfate for 16 h at 16°C. The enzymatic treatment was followed by 1-h incubation in calcium-free Barth's solution with gentle shaking for defolliculation. Defolliculated oocytes were incubated in the Barth's solution for 34 h before injection of 50 nl of CFTR cRNA (0.050.2 ng) or sterile water using a Nanoject autoinjector (Drummond Scientific, Broomall, PA). cRNA was prepared as previously described (6). Typically, oocytes were used 4896 h after injection.
Whole cell conductance and capacitance recordings.
Oocytes were placed in an oocyte recording chamber (model RC-3Z; Warner Instruments, Hamden, CT). Bathing solutions were changed using a gravity-driven perfusion system at a rate of 2 ml/min. The cells 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-97; Sutter Instruments, Novato, CA), filled with 3 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. Membrane conductance (Gm) and membrane capacitance (Cm) were recorded using a setup described by Weber et al. (36). In short, two digital signal processing (DSP) boards, which were equipped with two high-speed (300 kHz) A/D converters and two D/A converters, were connected to the two-electrode voltage-clamp amplifier via a multifunctional interface controlled by the DSP boards. One DSP board was used to record Gm, which was measured by imposing a sine wave (1 Hz frequency, 5 mV amplitude) to the clamped oocyte. Current changes evoked by the sine wave were sampled with a frequency of 625 Hz. Regression analysis was used to calculate macroscopic Gm from the current and voltage in a way that enabled the system to update conductance values every 10 s. The high-frequency Cm was measured with 146-Hz sine waves from the second board.
Single-channel recordings.
Single-channel recording protocols were adopted from Chan et al. (7) with some modifications. To remove the vitelline membrane, oocytes were shrunk for 10 min in stripping solution containing (in mM) 250 KAsp, 20 KCl, 1 MgCl2, 10 HEPES-KOH, and 1 EGTA, pH 7.4. The vitelline membrane was 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), and were 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) 138 N-methyl-D-glucammonium Cl, 2 MgCl2, 5 HEPES, and 0.1 GdCl3, titrated to pH 7.4 with HCl. Seals (150300 G
) were obtained by using gentle suction. The Ag-AgCl bath electrode was connected to the chamber bath using an agar bridge (2% agar in ND96). Outward unitary currents in CFTR channels were recorded at a pipette voltage (Vp) of 40 mV (Vp = 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 (pClamp 8.2; Axon Instruments). The digitized, baseline-corrected currents from recordings containing one to five channels were idealized using a segmental K-means algorithm (26). Parameters of amplitude histograms and event occupancy were used to calculate
and open probability (Po). Because of the CFTR outward rectification (2) in cell-attached patches, single-channel conductances were estimated from amplitude histograms of cell-attached recordings at holding voltages (Vh) of 20100 mV. In excised patches, the currents recorded at Vh 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 from recordings 210 min long using the equation (10)
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Drugs.
To activate PKA, we used a cAMP cocktail containing 250 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) and 25 µM forskolin (Sigma-Aldrich). To activate PKC, we used 250 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich). These treatments have maximal effects on CFTR conductance (6). Stock solutions of these compounds were prepared in water (8-BrcAMP), dimethyl sulfoxide (PMA), and ethanol (forskolin) and diluted to the desired final concentration in ND96 solution immediately before use. [2-(Trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET; Toronto Research Chemicals, North York, ON, Canada) was dissolved in bath solution to a 1 mM concentration immediately before use from a 1 M stock in water stored at 20°C. -Mercaptoethanol (Sigma-Aldrich) was dissolved in bath solution to a final 2 mM concentration immediately before use. At the concentrations used, the vehicles had no effects on CFTR conductance.
Statistical analysis. Given the known variability among batches of oocytes, all experiments were performed in at least three batches, and in every case, the results in all batches were in the same direction, although of varying magnitude. Summary data in the figures correspond to the batches with the highest number of experiments. Data are expressed as means ± SE. Differences between means were compared using 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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In Fig. 1, we show the effects of sequential exposure to PMA and cAMP cocktail, or vice versa, on the total conductance of oocytes expressing XCFTR. To obtain the current-voltage (I-V) plots, we applied a conventional step-voltage protocol before kinase stimulation and after the conductance approached a maximum. The increase in XCFTR conductance in response to PMA (comparing only first exposures) was approximately sixfold that of cAMP (Fig. 1, A and C; and Fig. 2C). This result differs from previous work at our laboratory (6) regarding the effects of cAMP and PMA on the same preparation. The earlier conclusion of our laboratory was therefore incorrect because the response to cAMP was assessed after exposure to PMA (see above). Hence, we conclude that the main difference between the responses of XCFTR and hCFTR to kinase activation is that XCFTR has a much smaller response than hCFTR to PKA stimulation.
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In conclusion, the effects of maximal PKA or PKC stimulation of hCFTR expressed in Xenopus oocytes are similar, whereas for XCFTR the response to PMA is approximately sixfold that to cAMP. Investigators at our laboratory (6) previously showed that differences in expression levels do not explain these results.
PKC activation does not cause insertion of new channels into the plasma membrane.
Changes in the total conductance of channels of one type in a cell membrane, at constant transmembrane electrochemical gradients, can result only from individual or combined effects on N (i.e., the number of active channels in the membrane), (i.e., the single-channel conductance), and Po. In this and the following sections, we summarize experiments designed to explore these three possibilities.
It has been reported that acute stimulation of PKA elicits an increase in Cm (Cm) of Xenopus oocytes expressing hCFTR (36). This observation was interpreted as an indication of an increase in the number of CFTR channels in the membrane, probably by exocytotic insertion. In our experiments, in parallel with the increase in conductance elicited by PMA or cAMP, we found that the apparent Cm also increased, especially in response to PMA. The
Cm in XCFTR stimulated by cAMP cocktail and PMA were 18 and 39%, respectively. The
Cm in hCFTR stimulated by PMA was 35%, which is not significantly different from the change in XCFTR (Fig. 3). However, membrane insertion does not necessarily parallel channel insertion. To address more directly whether there is CFTR insertion, we resorted to the substituted-cysteine accessibility method (SCAM) (1), previously applied to hCFTR by Liu et al. (20). This method is based on the introduction of single Cys residues at specific sites of a membrane protein. The cell expressing the Cys mutant is then exposed to a charged hydrophilic thiol reagent, and the functional effect of the reagent is assessed. In the case of an ion channel, a change in conductance indicates that the introduced Cys is inside the pore or at its mouth. In the present study, we used SCAM on CFTR channels expressed on the membrane by exposure to the thiol reagent MTSET, which reacts covalently with the Cys residue. After washing out the MTSET, we stimulated CFTR with a PKC agonist to determine whether a second exposure to MTSET after PKC stimulation would cause a new population (of unlabeled channels) to be inserted into the membrane. If this were the case, then a second exposure to the thiol reagent would alter the conductance; if no new channels were inserted, then the thiol reagent would have no effect.
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Changes in single-channel conductance do not account for the effect of PMA on XCFTR.
To investigate the mechanism of the changes in macroscopic conductance elicited by PMA and cAMP, we used the cell-attached patch-clamp technique. Because of the existence of endogenous oocyte Cl channels (35), four criteria were used to identify CFTR channels: 1) no or rare openings without kinase stimulation, 2) channel opening induced by PMA or cAMP, 3) between 6 and 11 pS, and 4) voltage independence of the Po. The results are illustrated in Figs. 5, 6, and 7 and summarized in Fig. 9A.
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The outward rectification of the XCFTR single-channel current to PMA stimulation in the cell-attached configuration is consistent with results obtained with the luminal membrane of acini of frog skin, one of the XCFTR endogenous expression sites (30), and also has been observed in hCFTR in cell-attached patches or in excised inside-out patches exposed to asymmetric Cl solutions (2, 14, 17, 23, 41). These observations have been attributed to permeant anion block and/or to Goldman-Hodgkin-Katz behavior (11).
Large changes in single-channel open probability account for the effects of cAMP and PMA on XCFTR. We determined the single-channel Po in cell-attached patches in oocytes expressing XCFTR and stimulated with either cAMP cocktail or PMA. Because of the gating properties of CFTR, only long recordings (210 min) were used for these studies. The results are illustrated in Fig. 8 and summarized in Fig. 9. The Po of cell-attached XCFTR channels at 40 mV was 0.05 ± 0.02 (n = 6) with cAMP cocktail stimulation and 0.23 ± 0.05 (n = 9) with PMA stimulation (Fig. 9B). In other words, the Po values differed approximately fivefold, a large, highly significant change (P < 0.001) that, given the variability of these measurements, accounts for the change in macroscopic conductance.
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Taken together, these results indicate that the dominant biophysical mechanism of PKC activation on XCFTR macroscopic conductance is an increase in Po with little, if any, effect on and N.
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DISCUSSION |
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PKC activation of CFTR.
PKC activation upregulates the apical membrane Cl conductance of epithelial cells (37), an effect observed in cells with both native and heterologous hCFTR expression (4, 12, 13, 16, 19, 21, 24, 38, 39). However, in contrast to the great effect of PKA stimulation, PKC agonists had a lesser effect (15). As shown in the present study, this is not the case for XCFTR, from which PKC stimulation elicits a response approximately sixfold that produced by PKA stimulation. In other words, the relative magnitudes of the effects of PKA and PKC stimulation differ between human and Xenopus CFTR, with the main difference being the much smaller response of XCFTR to cAMP. A nonspecific stimulatory effect of high PMA concentration can be ruled out because the same concentration of 4-PMA, an inactive form of PMA, had no effect, and the mutation T665A reduced the activation of XCFTR by PMA by
75% (6). In the present study, the activation of hCFTR by PKC stimulation is higher than that in other expression systems (4, 21, 40) but consistent with that previously reported for hCFTR expressed in Xenopus oocytes (39). The reasons for these differences are unknown.
There is no new channel insertion in response to PKC stimulation.
SCAM (1) was first used by Liu et al. (20) to address the question of hCFTR channel insertion into the plasma membrane in response to PKA stimulation in Xenopus oocytes. Their studies provided convincing evidence that this approach was sensitive in detecting both the channel insertion into the plasma membrane and that no appreciable CFTR channel insertion into the plasma membrane took place in response to hCFTR activation by PKA stimulation. These authors' results and conclusion are in apparent contradiction to the report that in the same experimental system, PKA stimulation caused an apparent Cm (36). In the present study, we used both techniques and found that PKC stimulation did elicit an apparent
Cm, but without insertion of new channels into the plasma membrane.
Cm in this kind of experiment are widely interpreted to indicate exocytosis, with proportional changes in membrane area, capacitance, and channel density. A serious problem exists in these studies in assessing changes in Cm when the conductance is increasing. In a recent, elegant study, careful correction for changes in Gm eliminated the apparent changes in Cm (9). In addition, even if the increase in capacitance were real, an increase in number of channels in the plasma membrane would occur only if the vesicles that fused to the membrane in response to kinase stimulation contained the channels in question, and proportionality between Gm and Cm changes would require, in addition, similar channel densities in the vesicles and the plasma membrane. Our results suggest that this is not the case. An important point must be made regarding the level of CFTR expression. High expression of CFTR has been proposed to saturate its trafficking, abrogating the
Cm in response to PKA stimulation (5, 32). The hCFTR expression level in Liu et al.'s study (20) was 510 times higher than that in Weber et al.'s study (36), assessed on the basis of Gm. On the basis of Gm in the present study, the expression of XCFTR was low, similar to the expression of hCFTR in Weber et al.'s work. In only one condition did we notice a small
Gm upon the second exposure to MTSET after PKC stimulation (
10% increase). This was observed 1 day after cRNA injection and suggests the possibility that there is a higher content of CFTR channels in subapical vesicles at this time. In summary, our data indicate that the contribution to the effect of PMA on Gm is minimal.
The results of the present studies apply only to CFTR expressed in Xenopus oocytes. The role of an increase in channel number in the membrane during the activation of CFTR by phosphorylation may be expression system dependent, as suggested by numerous other studies (for review, see Ref. 5) and also may depend on the status of the CFTR-trafficking process, as suggested by our work.
The activation of XCFTR channels by PKC stimulation is due to an increase in Po. The kinetics of XCFTR channels, i.e., bursts separated by long closures between bursts and flickery closures within the burst, is similar to that reported for hCFTR (2, 14, 17, 23, 41). The main effect of PKC stimulation on XCFTR at the single-channel level was a dramatic increase in Po, which largely accounts for the increase in macroscopic oocyte conductance. The Po when XCFTR was activated by a PKC agonist was similar to the Po value obtained by activation of hCFTR with a PKA agonist, yet much larger than the Po elicited by cAMP activation of XCFTR. This indicates that the main regulatory kinase of recombinant XCFTR expressed in Xenopus oocytes is PKC and not PKA.
The single-channel conductance (10.5 pS) in excised inside-out patches (in 140 mM Cl solutions on both sides) was voltage independent and of the same magnitude as the elicited by PKA stimulation of native XCFTR in frog skin glands (10.0 pS) (30) and of XCFTR expressed in HeLa cells (9.6 pS) (25). The conductance of XCFTR single channels activated by PKC in the cell-attached configuration was similar to that from inside-out patches, yet slightly higher than that elicited by PKA stimulation in the cell-attached patch. The difference was statistically significant but small and did not contribute significantly to the differences between the macroscopic conductances. To our knowledge, no differences in the
-values of hCFTR stimulated by either PKA or PKC agonists have been reported. If confirmed, the differences in single-channel conductances may indicate differences in dimension and/or charge of CFTR pores activated by either PKA or PKC stimulation. Further studies are needed to address this issue. Thus, under conditions of low-level membrane expression of XCFTR, the activation of XCFTR by stimulation of PKC does not involve net insertion of channels in the plasma membrane and is mostly attributable to an increase in Po.
In summary, the changes in macroscopic conductance and single-channel Po elicited by PKC-mediated stimulation of XCFTR expressed in Xenopus oocytes are similar to the responses of hCFTR to PKA stimulation, and the response of XCFTR to PKA stimulation is much smaller. This indicates that the hierarchies of the regulatory roles of PKA and PKC on human and Xenopus CFTR are opposite. Investigators at our laboratory previously found that most of the response of XCFTR to PKC depends on the consensus phosphorylation site containing T665 (6). The equivalent site is present in all of the CFTR orthologs for which sequences are available, from dogfish to chimpanzee, with the exception of zebrafish. Taken together, these findings raise interesting questions about the evolution of the CFTR molecule, in particular regarding the possibility that PKA-mediated phosphorylation is the dominant mechanism of CFTR regulation in only a few species, including humans.
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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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