Mechanism of activation of Xenopus CFTR by stimulation of PKC

Yongyue Chen, Guillermo A. Altenberg, and Luis Reuss

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was ~90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (~30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.

chloride channel; channel regulation; cystic fibrosis transmembrane conductance regulator gating; cystic fibrosis; phosphorylation; protein kinase A


THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) gene (18, 27, 28) codes for a phosphorylation- and ATP-dependent Cl channel expressed predominantly in the apical membrane of secreting epithelial cells, and also in other cells (15, 29). Gene mutations that alter the production, processing, regulation, or channel function of CFTR cause cystic fibrosis (29, 33). There is no doubt that PKA-mediated phosphorylation is the major regulatory mechanism of human CFTR (hCFTR) (3, 8, 34). However, recent data from our laboratory (6) regarding Xenopus CFTR (XCFTR) suggest that PKA- and PKC-mediated phosphorylation both activate XCFTR to a similar degree when expressed in either Xenopus oocytes or COS-1 cells. With regard to the effect of PKA stimulation, we found that the XCFTR response to PKC stimulation is much greater than that of hCFTR and that this difference depends on a single PKC phosphorylation consensus site in the regulatory (R) domain (T665LRR). A PKC consensus phosphorylation site at this location is found in all CFTR orthologs identified to date except the human one (T665LHR). The multisite, cooperative effect of PKA-mediated phosphorylation in the activation of CFTR (15, 29) and the single-site mechanism of the effect of PKC activation of XCFTR raise the possibility that the kinase effects are mediated by different molecular mechanisms. For these reasons, we decided to pursue studies to elucidate the biophysical mechanisms by which PKC stimulation increases XCFTR conductance.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
cDNA constructs and mutagenesis. The hCFTR DNA (gift from Dr. Lap-Chee Tsui, The Hospital for Sick Children, Toronto, ON, Canada) was cut with XmaI and SalI and cloned into the pOcyt7 plasmid (22) cut with the same restriction enzymes. The XCFTR DNA (gift from Drs. Margaret Price and Michael Welsh, University of Iowa, Iowa City, IA) was cloned into the KpnI and SalI sites of pOcyt7. XCFTR R334C was generated by substitution of Arg334 with Cys using the QuickChange multisite-directed mutagenesis kit (Stratagene, La Jolla, CA). The sequence of the mutagenic primer (mutant bases are underlined) was 5'-GCATTTCACTCTGTAAGATCT TTACTACCATTTCATTTAGC-3'. A silent BglII site was introduced for primary screening before DNA sequencing at the Protein Chemistry Laboratory of The University of Texas Medical Branch.

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 3–4 h before injection of 50 nl of CFTR cRNA (0.05–0.2 ng) or sterile water using a Nanoject autoinjector (Drummond Scientific, Broomall, PA). cRNA was prepared as previously described (6). Typically, oocytes were used 48–96 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 (22–24°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 0.5–1.5 M{Omega} 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 6–10 M{Omega} (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 (150–300 G{Omega}) 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 {gamma} 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 20–100 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 2–10 min long using the equation (10)

where Po is the open probability of a single channel, N is the number of channels in a patch, and OS is the occupancy time fraction at each current level (S = 1, 2, ..., N).

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. {beta}-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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The response of XCFTR to PKC stimulation is approximately sixfold that of PKA stimulation. In a previous study at our laboratory (6), PKC stimulation elicited 80% of the maximal activation obtained by simultaneous stimulation of PKA and PKC. The response of XCFTR to PKA stimulation was measured during exposure to PMA, a protocol based on the assumption that PKC stimulation does not significantly alter the response of CFTR to PKA agonists. Although this appears to be the case in cardiac CFTR (21, 24, 39), in the epithelial CFTR isoform, PKC stimulation potentiates the effect of PKA stimulation (31). More recent experiments performed at our laboratory revealed that PKC stimulation increased the subsequent response of XCFTR to PKA agonists (unpublished data). Therefore, to quantify correctly the responses of XCFTR to stimulation of either PKA or PKC, we considered only the first response of each oocyte to either cAMP cocktail or PMA and used only oocytes from the same batches.

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.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Activation of Xenopus cystic fibrosis transmembrane conductance regulator (XCFTR) channels expressed in Xenopus oocytes by PKC and PKA agonists. A and C show the time courses of XCFTR conductances, and B and D depict the current-voltage (I-V) plots at the peak conductance levels. Concentrations were phorbol 12-myristate 13-acetate (PMA), 250 nM; cAMP cocktail, 250 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), and 25 µM forskolin. A and B: response to PMA followed by response to cAMP cocktail. Exposure times are denoted by horizontal bars. C and D: response to cAMP cocktail followed by response to PMA.

 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. Activation of human CFTR (hCFTR) channels expressed in Xenopus oocytes by PMA and cAMP cocktail. A: response to cAMP cocktail followed by response to PMA. B: response to PMA followed by response to cAMP cocktail. C: summary of the responses of XCFTR (Fig. 1) and hCFTR (A and B) to PMA and cAMP cocktail (first exposure to either agent). The response of XCFTR to PMA was several times higher than its response to cAMP (P < 0.01; n = 6) and slightly higher than the response of hCFTR to cAMP (P < 0.05; n = 6).

 
The reversal potentials of the currents elicited by PMA and cAMP cocktail were similar (approximately –30 mV) (Fig. 1). Under the same experimental conditions, the conductance change of oocytes expressing hCFTR in response to PMA was indistinguishable from that elicited by cAMP cocktail (Fig. 2). Figs. 1 and 2 show that the responses of XCFTR and hCFTR to cAMP after transient exposure to PMA were significantly increased, with the effect on XCFTR being much larger (9a).

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), {gamma} (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 ({Delta}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 {Delta}Cm in XCFTR stimulated by cAMP cocktail and PMA were 18 and 39%, respectively. The {Delta}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.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3. Xenopus oocyte membrane capacitance (Cm) changes ({Delta}Cm) concomitant with the membrane conductance (Gm) changes ({Delta}Gm) elicited by exposure to cAMP cocktail or PMA. A and B: time courses of Cm and Gm responses of XCFTR-expressing oocytes to cAMP cocktail and PMA, respectively. C: {Delta}Cm and {Delta}Gm of oocytes expressing hCFTR in response to PMA. D: summary of apparent {Delta}Cm relative to baseline Cm. In XCFTR-expressing oocytes, the {Delta}Cm in response to PMA and cAMP were 39 ± 4% and 18 ± 2%, respectively (P < 0.01 in both series; n = 10). In hCFTR-expressing oocytes, the {Delta}Cm in response to PMA stimulation was 35 ± 4% (n = 6), which was not significantly different from that of XCFTR (P > 0.05).

 
Oocytes expressing XCFTR-R334C and wild-type XCFTR had similar responses to PMA. Exposure to 1 mM MTSET after stimulation with PMA increased the conductance by ~89% (Fig. 4, A and C). The response to MTSET was rapid (one-half the time required for the conductance increase, <20 s), sustained, and reversed by the reducing agent {beta}-mercaptoethanol (Fig. 4A). Similar results were reported by Liu et al. (20). MTSET (1 mM) had no effect on the conductance of oocytes expressing wild-type XCFTR in the absence or presence of PMA (data not shown). This indicates that MTSET modifies the channel conductance by reacting specifically with the introduced Cys at position 334. These results also indicate that this method has sufficient sensitivity to detect new channel insertion. As shown in Fig. 4, MTSET modifies the channel when it is open (Fig. 4A; increase in Gm at ~5 min) and when it is closed (Fig. 4B; preexposure prevents the effect of a second exposure).



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 4. Effects of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) on Gm of oocytes expressing XCFTR-R334C. A: after PMA stimulation, Gm was further increased by exposure to 1 mM MTSET for ~90 s. The effect of MTSET was rapid, sustained, and reversed by the reducing agent {beta}-mercaptoethanol ({beta}-ME; 1 mM). B: preexposure to MTSET blocked the increase in Gm in response to MTSET (for 15 s) after PMA stimulation. C: summary of Gm changes in response to MTSET. {Delta}Gm relative to the previous values were 89 ± 5% (n = 6) in the absence of pretreatment (first MTSET) and 5 ± 2% (n = 12) after pretreatment (second MTSET).

 
To test whether PMA stimulation causes insertion of new XCFTR channels, oocytes expressing the XCFTR-R334C mutant were first exposed to MTSET for 10–20 s, then the MTSET was thoroughly washed out and the oocytes were stimulated with PMA. When the increase in Gm approached a maximum, the oocytes were again exposed to MTSET. As illustrated in Fig. 4B and summarized in Fig. 4C, the second exposure to MTSET caused little or no change in Gm (i.e., the preexposure to MTSET largely prevented the Gm increase shown in Fig. 4A). This result indicates that no or very few new channels (i.e., channels not previously reacted with MTSET) were inserted into the membrane in response to PMA.

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) {gamma} 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.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. Cell-attached recordings of XCFTR single-channel activity elicited by PMA. A: channel openings in response to PMA stimulation at Vh = 40 mV. Second line, expanded recording of the marked interval; C, closed state. B: cell-attached current recordings at different Vh values. Outward rectification and flickering are evident at negative Vh. C: slope conductance at positive voltages was ~10 pS.

 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. Single XCFTR channel in an excised inside-out patch in symmetric Cl (140 mM) solution. A: XCFTR channel identified by PMA stimulation in a cell-attached patch was excised and exposed to symmetric Cl solutions. B: linear I-V plot. {gamma} = 10 pS. The {gamma} value of XCFTR in excised inside-out patches after PMA stimulation was 10.5 ± 0.2 pS (n = 3).

 


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7. Single-channel activity of XCFTR elicited by cAMP cocktail in a cell-attached patch. A: channel openings are stimulated by cAMP cocktail. Vh = 40 mV. B: I-V curve at positive Vh produces slope conductance of ~7.7 pS.

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 9. Summary of single-channel {gamma} and Po of XCFTR and hCFTR in cell-attached patches in response to stimulation by PKA and PKC agonists. A: single-channel {gamma} of XCFTR in response to kinase agonists in cell-attached patches were 9.9 ± 0.4 pS (n = 6) and 7.7 ± 0.5 pS (n = 5) for PMA and cAMP, respectively. Single-channel conductance of hCFTR in response to cAMP cocktail in cell-attached patches was 7.7 ± 0.6 pS (n = 4). B: Po of XCFTR in response to PMA and cAMP cocktail were 0.23 ± 0.02 (n = 9) and 0.05 ± 0.02 (n = 6), respectively. The Po of hCFTR in response to cAMP cocktail was 0.31 ± 0.02 (n = 4).

 
The kinetics of XCFTR in the cell-attached configuration (Fig. 5) resembled that of the excised inside-out patch (Fig. 6; see also Ref. 35). There were bursts separated by long closures and flickery closures within the bursts (Fig. 5A), a feature more evident at negative Vh values (Fig. 5B). The single-channel I-V relationship in the cell-attached patches demonstrated outward rectification with slope conductance (measured from the 20–100 mV interval) of 9.9 ± 0.4 pS (n = 6) and 7.7 ± 0.5 pS (n = 5) in response to PMA (Fig. 5C) and cAMP stimulation (Fig. 7B), respectively. The single-channel I-V relationship of inside-out patches excised after PKC stimulation in symmetric Cl solutions was linear, with {gamma} = 10.5 ± 0.2 pS (n = 3) (Fig. 6), a value similar to that reported for expression of XCFTR in HeLa cells (25). The mean {gamma}-value was 29% greater with PMA than with cAMP cocktail (P < 0.01). This difference accounts for only a minor part of the response to PMA vs. cAMP (Fig. 2C).

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 (2–10 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.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 8. XCFTR single-channel activity under stimulation by cAMP cocktail or PMA. A: cAMP-activated single-channel currents. Continuous recording in a cell-attached patch at Vh = 40 mV. The first trace illustrates the background channel activity before cAMP stimulation. B: PMA-activated single-channel current with protocol similar to that used in A. The recordings in A and B were from different oocytes.

 
The single-channel conductance and Po of hCFTR in the cell-attached configuration in response to cAMP stimulation were 7.7 ± 0.6 pS (n = 4) and 0.31 ± 0.02 (n = 4), respectively. These data, shown for comparison in Fig. 9, illustrate that in the oocyte expression system, the increase in Po by PKA stimulation of hCFTR is similar to that elicited by PKC stimulation of XCFTR.

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 {gamma} and N.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In trying to explain the difference between the responses of XCFTR to PKA and PKC stimulation, we reasoned that if the Po of XCFTR after PKA stimulation were similar to that of hCFTR (0.30–0.65) (7, 12), then the observed sixfold greater conductance increase with PKC stimulation would denote increases in {gamma} and/or N. To ascertain the biophysical mechanisms underlying the activation of XCFTR by PKC-mediated phosphorylation, we performed whole cell Gm measurements and cell-attached single-channel recordings with and without kinase stimulation. The results show that the activation of XCFTR by PKC stimulation occurs in the absence of channel insertion into the plasma membrane and is due to an approximately fivefold increase in Po compared with that elicited in XCFTR by PKA stimulation. Also, in response to PKA stimulation, the Po of XCFTR is much lower than the Po observed in hCFTR (2).

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{alpha}-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 {Delta}Cm (36). In the present study, we used both techniques and found that PKC stimulation did elicit an apparent {Delta}Cm, but without insertion of new channels into the plasma membrane. {Delta}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 {Delta}Cm in response to PKA stimulation (5, 32). The hCFTR expression level in Liu et al.'s study (20) was 5–10 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 {Delta}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 {gamma} 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 {gamma}-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.


    ACKNOWLEDGMENTS
 
We thank Dr. O. P. Hamill for help with the Xenopus oocyte single-channel experiments, Dr. M. J. Welsh and Dr. S. A. Weinman for discussions, and Lynette Durant for secretarial help.


    FOOTNOTES
 

Address for reprint requests and other correspondence: L. Reuss, Department of Neuroscience and Cell Biology, The Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0437lreuss{at}utmb.edu

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Akabas MH. Probing CFTR channel structure and function using the substituted-cysteine-accessibility method. Methods Mol Med 70: 159–174, 2002.[Medline]

2. Bear CE, Duguay F, Naismith AL, Kartner N, Hanrahan JW, and Riordan JR. Cl channel activity in Xenopus oocytes expressing the cystic fibrosis gene. J Biol Chem 266: 19142–19145, 1991.[Abstract/Free Full Text]

3. Bear CE, Li CH, Kartner N, Bridges RJ, Jensen TJ, Ramjeesingh M, and Riordan JR. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 68: 809–818, 1992.[ISI][Medline]

4. Berger HA, Travis SM, and Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl channel by specific protein kinases and protein phosphatases. J Biol Chem 268: 2037–2047, 1993.[Abstract/Free Full Text]

5. Bertrand CA and Frizzell RA. The role of regulated CFTR trafficking in epithelial secretion. Am J Physiol Cell Physiol 285: C1–C18, 2003.[Abstract/Free Full Text]

6. Button B, Reuss L, and Altenberg GA. PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site: insertion of this site confers PKC sensitivity to human CFTR. J Gen Physiol 117: 457–468, 2001.[Abstract/Free Full Text]

7. Chan KW, Csanády L, Seto-Young D, Nairn AC, and Gadsby DC. Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator's NH2-terminal nucleotide binding domain. J Gen Physiol 116: 163–180, 2000.[Abstract/Free Full Text]

8. Chang XB, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, Hanrahan JW, and Riordan JR. Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. J Biol Chem 268: 11304–11311, 1993.[Abstract/Free Full Text]

9. Chen P, Wang TC, and Gillis KD. The relationship between cAMP, Ca2+, and transport of CFTR to the plasma membrane. J Gen Physiol 118: 135–144, 2001.[Abstract/Free Full Text]

9. Chen Y, Button B, Altenberg GA, and Reuss L. Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role on NBD2. Am J Physiol Cell Physiol 287: C1436–C1444, 2004.[Abstract/Free Full Text]

10. Collier ML and Hume JR. Unitary chloride channels activated by protein kinase C in guinea pig ventricular myocytes. Circ Res 76: 317–324, 1995.[Abstract/Free Full Text]

11. Dawson DC, Smith SS, and Mansoura MK. CFTR: mechanism of anion conduction. Physiol Rev 79, Suppl: S47–S75, 1999.[Medline]

12. Dechecchi MC, Tamanini A, Berton G, and Cabrini G. Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. J Biol Chem 268: 11321–11325, 1993.[Abstract/Free Full Text]

13. Fischer H, Illek B, and Machen TE. Regulation of CFTR by protein phosphatase 2B and protein kinase C. Pflügers Arch 436: 175–181, 1998.[CrossRef][ISI][Medline]

14. Fischer H and Machen TE. CFTR displays voltage dependence and two gating modes during stimulation. J Gen Physiol 104: 541–566, 1994.[Abstract]

15. Gadsby DC and Nairn AC. Regulation of CFTR Cl ion channels by phosphorylation and dephosphorylation. Adv Second Messenger Phosphoprotein Res 33: 79–106, 1999.[ISI][Medline]

16. Jia Y, Mathews CJ, and Hanrahan JW. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J Biol Chem 272: 4978–4984, 1997.[Abstract/Free Full Text]

17. Kartner N, Hanrahan JW, Jensen TJ, Naismith AL, Sun SZ, Ackerley CA, Reyes EF, Tsui LC, Rommens JM, Bear CE, and Riordan JR. Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance. Cell 64: 681–691, 1991.[ISI][Medline]

18. Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, and Tsui LC. Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073–1080, 1989.[ISI][Medline]

19. Liedtke CM, Yun CH, Kyle N, and Wang D. Protein kinase C{epsilon}-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor. J Biol Chem 277: 22925–22933, 2002.[Abstract/Free Full Text]

20. Liu X, Smith SS, Sun F, and Dawson DC. CFTR: covalent modification of cysteine-substituted channels expressed in Xenopus oocytes shows that activation is due to the opening of channels resident in the plasma membrane. J Gen Physiol 118: 433–446, 2001.[Abstract/Free Full Text]

21. Middleton LM and Harvey RD. PKC regulation of cardiac CFTR Cl channel function in guinea pig ventricular myocytes. Am J Physiol Cell Physiol 275: C293–C302, 1998.[Abstract/Free Full Text]

22. Mo L, Hellmich HL, Fong P, Wood T, Embesi J, and Wills NK. Comparison of amphibian and human ClC-5: similarity of functional properties and inhibition by external pH. J Membr Biol 168: 253–264, 1999.[CrossRef][ISI][Medline]

23. Nagel G, Hwang TC, Nastiuk KL, Nairn AC, and Gadsby DC. The protein kinase A-regulated cardiac Cl channel resembles the cystic fibrosis transmembrane conductance regulator. Nature 360: 81–84, 1992.[CrossRef][ISI][Medline]

24. Picciotto MR, Cohn JA, Bertuzzi G, Greengard P, and Nairn AC. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 267: 12742–12752, 1992.[Abstract/Free Full Text]

25. Price MP, Ishihara H, Sheppard DN, and Welsh MJ. Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR. J Biol Chem 271: 25184–25191, 1996.[Abstract/Free Full Text]

26. Qin F, Auerbach A, and Sachs F. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys J 70: 264–280, 1996.[Abstract]

27. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FS, and Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073, 1989.[ISI][Medline]

28. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N, Zsiga M, Buchwald M, Riordan JR, Tsui LC, and Collins FS. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059–1065, 1989.[ISI][Medline]

29. Sheppard DN and Welsh MJ. Structure and function of the CFTR chloride channel. Physiol Rev 79, Suppl: S23–S45, 1999.[Medline]

30. Sørensen JB and Larsen EH. Patch clamp on the luminal membrane of exocrine gland acini from frog skin (Rana esculenta) reveals the presence of cystic fibrosis transmembrane conductance regulator-like Cl channels activated by cyclic AMP. J Gen Physiol 112: 19–31, 1998.[Abstract/Free Full Text]

31. Tabcharani JA, Chang XB, Riordan JR, and Hanrahan JW. Phosphorylation-regulated Cl channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352: 628–631, 1991.[CrossRef][ISI][Medline]

32. Takahashi A, Watkins SC, Howard M, and Frizzell RA. CFTR-dependent membrane insertion is linked to stimulation of the CFTR chloride conductance. Am J Physiol Cell Physiol 271: C1887–C1894, 1996.[Abstract/Free Full Text]

33. Tsui LC. The cystic fibrosis transmembrane conductance regulator gene. Am J Respir Crit Care Med 151: S47–S53, 1995.[ISI][Medline]

34. Wang X, Marunaka Y, Fedorko L, Dho S, Foskett JK, and O'Brodovich H. Activation of Cl currents by intracellular chloride in fibroblasts stably expressing the human cystic fibrosis transmembrane conductance regulator. Can J Physiol Pharmacol 71: 645–649, 1993.[ISI][Medline]

35. Weber WM. Endogenous ion channels in oocytes of Xenopus laevis: recent developments. J Membr Biol 170: 1–12, 1999.[CrossRef][ISI][Medline]

36. Weber WM, Cuppens H, Cassiman JJ, Clauss W, and Van Driessche W. Capacitance measurements reveal different pathways for the activation of CFTR. Pflügers Arch 438: 561–569, 1999.[CrossRef][ISI][Medline]

37. Welsh MJ, Li M, McCann JD, Clancy JP, and Anderson MP. Phosphorylation-dependent regulation of apical membrane chloride channels in normal and cystic fibrosis airway epithelium. Ann NY Acad Sci 574: 44–51, 1989.[Abstract]

38. Winter MC, Sheppard DN, Carson MR, and Welsh MJ. Effect of ATP concentration on CFTR Cl channels: a kinetic analysis of channel regulation. Biophys J 66: 1398–1403, 1994.[Abstract]

39. Yamazaki J, Britton F, Collier ML, Horowitz B, and Hume RR. Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C. Biophys J 76: 1972–1987, 1999.[Abstract/Free Full Text]

40. Yurko-Mauro KA and Reenstra WW. Prostaglandin F2{alpha} stimulates CFTR activity by PKA- and PKC-dependent phosphorylation. Am J Physiol Cell Physiol 275: C653–C660, 1998.[Abstract]

41. Zhou Z, Hu S, and Hwang TC. Voltage-dependent flickery block of an open cystic fibrosis transmembrane conductance regulator (CFTR) channel pore. J Physiol 532: 435–448, 2001.[Abstract/Free Full Text]