| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
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
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) was increased by 
22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH2-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in Po (with a small change in ) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains. 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, 36–38, 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.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
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 (22–24°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 0.5–1.5 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 6–10 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 20–100 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):
 |
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.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
. First, we addressed the possibility that exocytic insertion of new channels from an intracellular pool is responsible, solely or in part, for the potentiation effect in XCFTR. To this end, we used the substituted-cysteine-accessibility method (1, 2, 24). Arg334, a position thought to be in the extracellular mouth of the CFTR channel and accessible from the extracellular side, was mutated to Cys. Charged, membrane-impermeable thiol reagents react at this site and change the conductance of the CFTR channels (1, 24). In control experiments, the increase in Gm elicited by the thiol reagent MTSET+ (1 mM) was 98 ± 5% (n = 6) in activated XCFTR-R334C (Fig. 4, A and B). The effect of MTSET+ was persistent but could be fully reversed by the reducing agent -ME. MTSET+ had no effect on wild-type CFTR (data not shown). To test for channel insertion, oocytes expressing XCFTR-R334C were exposed to MTSET+ for 10–20 s during the first PKA stimulation and then the agent was washed out from the bath. The oocyte was then stimulated by PKC and PKA sequentially and reexposed to MTSET+ when the conductance change reached a plateau after the second PKA stimulation (Fig. 4, A and B). Under these conditions, any new channels inserted in the membrane by the action of the kinases would be responsive to the second exposure to MTSET+, whereas the preexisting channels would not, because they remain labeled by the preexposure to the MTSET+. As illustrated in Fig. 4C, when oocytes expressing XCFTR-R334C were stimulated by PKA subsequent to PKA and PKC stimulation, there was no appreciable change in conductance with the second exposure to MTSET+. These results, summarized in Fig. 4D, rule out a measurable role of a change in N as an explanation for the PKC potentiation effect on PKA activation of XCFTR.
|
on the macroscopic CFTR conductance elicited by the potentiation effect, we used the cell-attached patch-clamp technique. Detailed single XCFTR channel properties in the cell-attached configuration in Xenopus oocytes are described by us elsewhere (13). The gating pattern (Fig. 5A) of XCFTR expressed in Xenopus oocytes resembled that observed in cells with endogenous expression (39). Single-channel slope conductance after PKA stimulation was 7.7 ± 0.5 pS, and Po was 0.05 ± 0.02 (13). After PKC stimulation, PKA stimulation elicited openings with a of 9.4 ± 0.4 pS (n = 5; Fig. 5B) and a Po of 0.14 ± 0.03 (n = 6; Fig. 5C). Both differences were statistically significant; at the P < 0.025 level and Po at the P < 0.001 level, indicating that both Po and during PKA stimulation are increased by preexposure to PKC agonists. The magnitudes of the changes are very different, however. Together, the average increases ( by 22% and Po by 180%) should yield an increase in Gm by 3.5-fold, most of which is attributable to the increase in Po (see DISCUSSION).
|
|
|
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).
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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 Hanrahan’s 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 Dawson’s 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 Dawson’s 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.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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 |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2. Akabas MH, Cheung M, and Guinamard R. Probing the structural and functional domains of the CFTR chloride channel. J Bioenerg Biomembr 29: 453–463, 1997.
3. Anderson MP, Rich DP, Gregory RJ, Smith AE, and Welsh MJ. Generation of cAMP-activated chloride currents by expression of CFTR. Science 251: 679–682, 1991.
4. Baldursson O, Berger HA, and Welsh MJ. Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia. Am J Physiol Lung Cell Mol Physiol 279: L835–L841, 2000.
5. 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.
6. 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.
7. 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.
8. Chan KW, Csanady 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.
9. 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.
10. Chappe V, Hinkson DA, Howell LD, Evagelidis A, Liao J, Chang XB, Riordan JR, and Hanrahan JW. Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 101: 390–395, 2004.
11. Chappe V, Hinkson DA, Zhu T, Chang XB, Riordan JR, and Hanrahan JW. Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA. J Physiol 548: 39–52, 2003.
12. Chen P, Hwang TC, and Gillis KD. The relationship between cAMP, Ca2+, and transport of CFTR to the plasma membrane. J Gen Physiol 118: 135–144, 2001.
13. Chen Y, Altenberg GA, and Reuss L. Mechanisms of the activation of Xenopus CFTR by stimulation of PKC. Am J Physiol Cell Physiol 287: C1256–C1263, 2004.
14. Cheng SH, Rich DP, Marshall J, Gregory RJ, Welsh MJ, and Smith AE. Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66: 1027–1036, 1991.
15. Collier ML and Hume JR. Unitary chloride channels activated by protein kinase C in guinea pig ventricular myocytes. Circ Res 76: 317–324, 1995.
16. 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.
17. Denning GM, Ostedgaard LS, Cheng SH, Smith AE, and Welsh MJ. Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia. J Clin Invest 89: 339–349, 1992.
18. Derkach V, Barria A, and Soderling TR. Ca2+/calmodulin-kinase II enhances channel conductance of 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proc Natl Acad Sci USA 96: 3269–3274, 1999.
19. Dulhanty AM and Riordan JR. Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. Biochemistry 33: 4072–4079, 1994.
20. French PJ, Bijman J, Edixhoven M, Vaandrager AB, Scholte BJ, Lohmann SM, Nairn AC, and de Jonge HR. Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II. J Biol Chem 270: 26626–26631, 1995.
21. Gadsby DC and Nairn AC. Regulation of CFTR Cl– ion channels by phosphorylation and dephosphorylation. Adv Second Messenger Phosphoprotein Res 33: 79–106, 1999.
22. Ganeshan R, Di A, Nelson DJ, Quick MW, and Kirk KL. The interaction between syntaxin 1A and cystic fibrosis transmembrane conductance regulator Cl– channels is mechanistically distinct from syntaxin 1A-SNARE interactions. J Biol Chem 31: 2876–2885, 2003.
23. 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.
24. 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.
25. Loffing J, Moyer BD, McCoy D, and Stanton BA. Exocytosis is not involved in activation of Cl– secretion via CFTR in Calu-3 airway epithelial cells. Am J Physiol Cell Physiol 275: C913–C920, 1998.
26. Loo DD, Hirsch JR, Sarkar HK, and Wright EM. Regulation of the mouse retinal taurine transporter (TAUT) by protein kinases in Xenopus oocytes. FEBS Lett 392: 250–254, 1996.
27. Ma J, Zhao J, Drumm ML, Xie J, and Davis PB. Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel. J Biol Chem 272: 28133–28141, 1997.
28. McCray PB Jr, Bettencourt JD, Bastacky J, Denning GM, and Welsh MJ. Expression of CFTR and a cAMP-stimulated chloride secretory current in cultured human fetal alveolar epithelial cells. Am J Respir Cell Mol Biol 9: 578–585, 1993.
29. Moreno AP, Saez JC, Fishman GI, and Spray DC. Human connexin43 gap junction channels. Regulation of unitary conductances by phosphorylation. Circ Res 74: 1050–1057, 1994.
30. Nishizaki T and Sumikawa K. Nicotinic receptors are regulated by protein kinase C activated via a nicotinic receptors-mediated signaling pathway. Brain Res Mol Brain Res 61: 211–218, 1998.
31. Ostedgaard LS, Baldursson O, and Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl– channel by its R domain. J Biol Chem 276: 7689–7692, 2001.
32. 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.
33. 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.
34. Reddy MM and Quinton PM. Deactivation of CFTR-Cl conductance by endogenous phosphatases in the native sweat duct. Am J Physiol Cell Physiol 270: C474–C480, 1996.
35. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, and Chou JL. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073, 1989.
36. Rommens JM, Dho S, Bear CE, Kartner N, Kennedy D, Riordan JR, Tsui LC, and Foskett JK. cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 88: 7500–7504, 1991.
37. Sheppard DN, Carson MR, Ostedgaard LS, Denning GM, and Welsh MJ. Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium. Am J Physiol Lung Cell Mol Physiol 266: L405–L413, 1994.
38. Sheppard DN and Welsh MJ. Structure and function of the CFTR chloride channel. Physiol Rev 79: S23–S45, 1999.
39. 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.
40. 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.
41. Tabcharani JA, Linsdell P, and Hanrahan JW. Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels. J Gen Physiol 110: 341–354, 1997.
42. 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.
43. Tsui LC. The cystic fibrosis transmembrane conductance regulator gene. Am J Respir Crit Care Med 151: S47–S53, 1995.
44. Wang S, Yue H, Derin RB, Guggino WB, and Li M. Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell 103: 169–179, 2000.
45. 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.
46. Winter MC and Welsh MJ. Stimulation of CFTR activity by its phosphorylated R domain. Nature 389: 294–296, 1997.
This article has been cited by other articles:
|
|
 |
|
  R Wimmer, S Hohenester, T Pusl, G U Denk, C Rust, and U Beuers Tauroursodeoxycholic acid exerts anticholestatic effects by a cooperative cPKC{alpha}-/PKA-dependent mechanism in rat liver Gut, October 1, 2008; 57(10): 1448 - 1454. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Alessandri-Haber, O. A. Dina, E. K. Joseph, D. Reichling, and J. D. Levine A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators. J. Neurosci., April 5, 2006; 26(14): 3864 - 3874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen, G. A. Altenberg, and L. Reuss Mechanism of activation of Xenopus CFTR by stimulation of PKC Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1256 - C1263. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
Gm) normalized to the conductance before MTSET (Gm) (n = 6). The first MTSET exposure elicits a large change in Gm (0.98 ± 0.10), whereas the second exposure has a much smaller effect (0.05 ± 0.02). The difference (paired analysis) was significant at P < 0.01.
