Regulation of CFTR Trafficking by Its R Domain
2008; Elsevier BV; Volume: 283; Issue: 42 Linguagem: Inglês
10.1074/jbc.m800516200
ISSN1083-351X
AutoresChristopher Lewarchik, Kathryn W. Peters, Juanjuan Qi, Raymond A. Frizzell,
Tópico(s)Ion channel regulation and function
ResumoPhosphorylation of the R domain is required for cystic fibrosis transmembrane conductance regulator (CFTR) channel gating, and cAMP/protein kinase A (PKA) simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments (Bertrand, C. A., and Frizzell, R. A. (2003) Am. J. Physiol. 285, C1–C18). We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R domain (Δamino acids 635–834) produced a channel with elevated basal current (Im) and no ΔIm or trafficking response (ΔCm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817–838, a 22-amino acid conserved helical region having a net charge of -9, termed NEG2 (Xie, J., Adams, L. M., Zhao, J., Gerken, T. A., Davis, P. B., and Ma, J. (2002) J. Biol. Chem. 277, 23019–23027), produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. Injection of NEG2 peptides into oocytes expressing split ΔNEG2 CFTR prior to stimulation restored the agonist-evoked ΔCm, consistent with the concept that this sequence mediates the regulated trafficking event. In support of this idea, ΔNEG2 CFTR escaped from the inhibition of wild type CFTR trafficking produced by overexpression of syntaxin 1A. These observations suggest that the NEG2 region at the C terminus of the R domain allows stabilization of CFTR in a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA stimulation. Phosphorylation of the R domain is required for cystic fibrosis transmembrane conductance regulator (CFTR) channel gating, and cAMP/protein kinase A (PKA) simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments (Bertrand, C. A., and Frizzell, R. A. (2003) Am. J. Physiol. 285, C1–C18). We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R domain (Δamino acids 635–834) produced a channel with elevated basal current (Im) and no ΔIm or trafficking response (ΔCm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817–838, a 22-amino acid conserved helical region having a net charge of -9, termed NEG2 (Xie, J., Adams, L. M., Zhao, J., Gerken, T. A., Davis, P. B., and Ma, J. (2002) J. Biol. Chem. 277, 23019–23027), produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. Injection of NEG2 peptides into oocytes expressing split ΔNEG2 CFTR prior to stimulation restored the agonist-evoked ΔCm, consistent with the concept that this sequence mediates the regulated trafficking event. In support of this idea, ΔNEG2 CFTR escaped from the inhibition of wild type CFTR trafficking produced by overexpression of syntaxin 1A. These observations suggest that the NEG2 region at the C terminus of the R domain allows stabilization of CFTR in a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA stimulation. The cystic fibrosis transmembrane conductance regulator (CFTR) 2The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel; IBMX, isobutylmethylxanthine; PKA, protein kinase A; WT, wild type; F, farad; HRP, horseradish peroxidase; NBD, nucleotide-binding domain; TMD, transmembrane domain; EXT, extope; HA, hemagglutinin; BFA, brefeldin A; ABC, ATP-binding cassette; sNEG2, scrambled NEG2; hNEG2, helical NEG2. 2The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel; IBMX, isobutylmethylxanthine; PKA, protein kinase A; WT, wild type; F, farad; HRP, horseradish peroxidase; NBD, nucleotide-binding domain; TMD, transmembrane domain; EXT, extope; HA, hemagglutinin; BFA, brefeldin A; ABC, ATP-binding cassette; sNEG2, scrambled NEG2; hNEG2, helical NEG2. is a phosphorylation-activated anion channel located at the apical membranes of airway, intestinal, pancreatic, and salivary gland epithelial cells. Its stimulation, primarily by cAMP-dependent signaling pathways, is the basis of electrolyte and fluid secretion that provides the fluid vehicle for macromolecular secretory products. In airway epithelia, CFTR is the principal apical anion conductance contributing to chloride and HCO3 secretion, and this establishes the electrical and osmotic driving forces for secondary sodium and water transport. Together, these events regulate the volume and composition of the airway surface liquid (3Chambers L. Rollins B. Tarran R. Respir. Physiol. Neurobiol. 2007; 159: 256-270Crossref PubMed Scopus (69) Google Scholar, 4Pilewski J. Frizzell R. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (379) Google Scholar). Mutations in the gene encoding CFTR cause cystic fibrosis by either reducing its apical membrane density or interfering with its ability to transport anions (4Pilewski J. Frizzell R. Physiol. Rev. 1999; 79: S215-S255Crossref PubMed Scopus (379) Google Scholar). Identification of the primary amino acid sequence (5Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. et al.Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5802) Google Scholar) placed CFTR in the ATP-binding cassette (ABC) transporter superfamily, of which there are ∼50 members in the human genome. Similar to other ABC transporters (6Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3321) Google Scholar), the N terminus of CFTR leads to six membrane-spanning segments that comprise the first transmembrane domain (TMD1), followed by a nucleotide-binding domain (NBD1). These structural elements are repeated in the C-terminal half of CFTR, as TMD2 and NBD2, followed by a C-terminal tail. A unique feature of CFTR among ABC family members is the presence of a regulatory (R) domain, interposed between these repeated TMD-NBD elements, whose multiple phosphorylation sites mediate cAMP-dependent channel activation by protein kinase A (PKA) (7Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79: S77-S107Crossref PubMed Scopus (364) Google Scholar). The multiple PKA phosphorylation sites of the R domain act in concert to enable gating, and site mutagenesis has not revealed a requirement for phosphorylation at specific loci (8Chang X.B. Tabcharani J.A. Hou Y.X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar). Rather, there is redundancy in the ability of R domain PKA sites to support channel activation. The unstructured nature of the R domain (9Ostedgaard L.S. Baldursson O. Vermeer D.W. Welsh M.J. Robertson A.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5657-5662Crossref PubMed Scopus (92) Google Scholar) was confirmed in recent NMR structural studies, which showed that this largely disordered region contains segments of helical structure that likely interact with other CFTR domains, and perhaps other proteins, to effect its regulatory functions (10Baker J.M. Hudson R.P. Kanelis V. Choy W.Y. Thibodeau P.H. Thomas P.J. Forman-Kay J.D. Nat. Struct. Mol. Biol. 2007; 14: 738-745Crossref PubMed Scopus (220) Google Scholar). Phosphorylation of the R domain is required for channel gating, which is then driven by the binding and hydrolysis of ATP at the NBDs of CFTR (11Anderson M.P. Berger H.A. Rich D.P. Gregory R.J. Smith A.E. Welsh M.J. Cell. 1991; 67: 775-784Abstract Full Text PDF PubMed Scopus (406) Google Scholar, 12Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (518) Google Scholar, 13Tabcharani J.A. Chang X.B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (451) Google Scholar, 14Vergani P. Lockless S.W. Nairn A.C. Gadsby D.C. Nature. 2005; 433: 876-880Crossref PubMed Scopus (329) Google Scholar). The formation of a head-to-tail NBD1/NBD2 dimer is thought to create shared ATP-binding sites that are contributed by residues from both NBDs, an arrangement based on bacterial ABC transporter structures (15Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). Conversely, the reversal of PKA-mediated channel activation requires R domain dephosphorylation, which is facilitated by phosphatases 2A and 2C (16Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274: C1397-C1410Crossref PubMed Google Scholar). The current view of R domain regulation of channel gating involves both inhibitory and stimulatory properties of this region. Under nonstimulated conditions, channel gating is inhibited by R domain elements that perhaps interfere with NBD dimer formation. This model is supported by the finding that addition of the nonphosphorylated R domain to the cytoplasmic face of active CFTR channels is inhibitory (17Ma J. Zhao J. Drumm M.L. Xie J. Davis P.B. J. Biol. Chem. 1997; 272: 28133-28141Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Conversely, a stimulatory action of the R domain was implicated by the properties of CFTR channels bearing a large R domain deletion (708–835), which gate with reduced open probability, but are stimulated by the addition of a phosphorylated R region polypeptide (amino acids 645–835) (18Winter M.C. Welsh M.J. Nature. 1997; 389: 294-296Crossref PubMed Scopus (124) Google Scholar). Finally, it appears that the location of the R domain, at the center of the repeated ABC structure, is not critical to channel regulation, because its transplantation to the C terminus of CFTR, together with a sufficient linker region, also conferred regulated CFTR channel function (19Baldursson O. Ostedgaard L.S. Rokhlina T. Cotten J.F. Welsh M.J. J. Biol. Chem. 2001; 276: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In addition to regulating channel gating, cAMP agonists also modulate the density of CFTR channels in the plasma membrane in many systems (for review see Ref. 1Bertrand C.A. Frizzell R.A. Am. J. Physiol. 2003; 285: C1-C18Crossref PubMed Scopus (114) Google Scholar). Accordingly, the cAMP/PKA-induced increase in anion current associated with phosphorylation-dependent increases in CFTR open probability (Po) is supported also by an increase in the number of channels (N) resident in the surface membrane. The influence of CFTR on membrane trafficking was first demonstrated in cystic fibrosis pancreatic cells in which the exogenous expression of WT CFTR produced a cAMP-dependent inhibition of endocytosis and stimulated the return of internalized membrane to the cell surface (i.e. recycling) (20Bradbury N.A. Jilling T. Berta G. Sorscher E.J. Bridges R.J. Kirk K.L. Science. 1992; 256: 530-532Crossref PubMed Scopus (294) Google Scholar). Similar results were obtained from human airway cells endogenously expressing WT CFTR, in which cAMP stimulation increased the release of previously internalized fluorescein isothiocyanate-dextran (21Schwiebert E.M. Gesek F. Ercolani L. Wjasow C. Gruenert D.C. Karlson K. Stanton B.A. Am. J. Physiol. 1994; 267: C272-C281Crossref PubMed Google Scholar). This exocytic event coincided with an increase in membrane capacitance, and these responses were present only in cells expressing WT CFTR. In addition, the kinetics of CFTR trafficking were relatively rapid. Following biotinylation of the cell surface, CFTR was internalized at rates that approached those of endocytic model proteins, such as the transferrin receptor (22Lukacs G.L. Segal G. Kartner N. Grinstein S. Zhang F. Biochem. J. 1997; 328: 353-361Crossref PubMed Scopus (119) Google Scholar, 23Prince L.S. Workman Jr., R.B. Marchase R.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5192-5196Crossref PubMed Scopus (119) Google Scholar). In Xenopus oocytes, the transit of CFTR to the cell surface, detected with an external epitope-tagged CFTR construct, paralleled acute cAMP/PKA-induced increases in membrane current and capacitance, and these functional responses were not observed in the absence of CFTR expression (24Takahashi A. Watkins S.C. Howard M. Frizzell R.A. Am. J. Physiol. 1996; 271: C1887-C1894Crossref PubMed Google Scholar, 25Peters K.W. Qi J. Watkins S.C. Frizzell R.A. Am. J. Physiol. 1999; 277: C174-C180Crossref PubMed Google Scholar). Importantly, CFTR immunolocalization data obtained with native tissues endogenously expressing CFTR provided data consistent with its agonist-evoked trafficking to the apical plasma membrane (26Ameen N.A. Marino C. Salas P.J. Am. J. Physiol. 2003; 284: C429-C438Crossref PubMed Scopus (35) Google Scholar, 27Lehrich R.W. Aller S.G. Webster P. Marino C.R. Forrest Jr., J.N. J. Clin. Investig. 1998; 101: 737-745Crossref PubMed Scopus (82) Google Scholar) Thus, biochemical, morphological, and functional evidence of regulated CFTR trafficking has been presented for a number of epithelial and nonepithelial systems (1Bertrand C.A. Frizzell R.A. Am. J. Physiol. 2003; 285: C1-C18Crossref PubMed Scopus (114) Google Scholar). Nevertheless, data conflicting with this concept have emerged as well. Although some negative findings can be attributed to the use of exogenous overexpression systems or nonphysiological experimental conditions (reviewed in Ref. 1Bertrand C.A. Frizzell R.A. Am. J. Physiol. 2003; 285: C1-C18Crossref PubMed Scopus (114) Google Scholar), it is evident also that an appropriate cellular background is needed to support regulated CFTR trafficking processes. Context dependence of cellular trafficking events is observed not only for CFTR, but for other channels and transporters (1Bertrand C.A. Frizzell R.A. Am. J. Physiol. 2003; 285: C1-C18Crossref PubMed Scopus (114) Google Scholar). Thus, the ability of agonists to alter plasma membrane CFTR density varies among cell types, likely because of variable expression of the required trafficking machinery and interacting proteins. However, the discovery that disease-causing mutations influence plasma membrane CFTR density by affecting its trafficking in distal secretory and recycling compartments (28Lukacs G.L. Chang X.B. Bear C. Kartner N. Mohamed A. Riordan J.R. Grinstein S. J. Biol. Chem. 1993; 268: 21592-21598Abstract Full Text PDF PubMed Google Scholar, 29Silvis M.R. Picciano J.A. Bertrand C. Weixel K. Bridges R.J. Bradbury N.A. J. Biol. Chem. 2003; 278: 11554-11560Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) highlights the significance of these processes and their contribution to normal epithelial functions. The inability of the common CFTR mutant to recycle to the plasma membrane following its internalization is a significant factor complicating the therapeutic rescue of ΔF508 CFTR to the cell surface (28Lukacs G.L. Chang X.B. Bear C. Kartner N. Mohamed A. Riordan J.R. Grinstein S. J. Biol. Chem. 1993; 268: 21592-21598Abstract Full Text PDF PubMed Google Scholar, 30Heda G.D. Tanwani M. Marino C.R. Am. J. Physiol. 2001; 280: C166-C174Crossref PubMed Google Scholar, 31Sharma M. Benharouga M. Hu W. Lukacs G.L. J. Biol. Chem. 2001; 276: 8942-8950Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The trafficking pathways and associated protein interactions that underlie regulated CFTR trafficking remain poorly defined. Prior studies have indicated that the CFTR trafficking response is robust in Xenopus oocytes expressing WT CFTR, demonstrated by parallel increases in membrane conductance (Gm) and capacitance (Cm) in response to cAMP/PKA stimulation (32Weber W.M. Cuppens H. Cassiman J.J. Clauss W. Van Driessche W. Pfluegers Arch. 1999; 438: 561-569Crossref PubMed Scopus (46) Google Scholar). These findings were supported also by increased cell surface detection of CFTR bearing an external epitope tag (25Peters K.W. Qi J. Watkins S.C. Frizzell R.A. Am. J. Physiol. 1999; 277: C174-C180Crossref PubMed Google Scholar), and increases in plasma membrane CFTR density were detected using atomic force microscopy applied to membranes from oocytes expressing CFTR (33Schillers H. Danker T. Madeja M. Oberleithner H. J. Membr. Biol. 2001; 180: 205-212Crossref PubMed Scopus (27) Google Scholar). These findings, together with the unitary relationship between the surface area and the electrical capacitance of biological membranes (1 μF/cm2), are consistent with the concept that cAMP/PKA stimulation elicits the fusion of CFTR-containing membranes from intracellular compartments with the plasma membrane. In this study, we examined the structural basis of regulated CFTR trafficking using a series of CFTR deletion constructs, by assessing their cAMP/PKA-induced membrane current and capacitance responses. Our goal was to determine whether there are regions of CFTR required for its regulated trafficking. In addition to producing a nonregulated chloride current in the absence of stimulation, we found that removal of the R domain eliminated the cAMP/PKA-induced membrane capacitance increase. Further structure-function analysis of R domain deletion constructs showed that removal of its C-terminal region produced a channel that retained agonist-dependent current regulation, but eliminated its cAMP/PKA-mediated trafficking, as reflected by the agonist-induced increase in Cm. These findings suggest that a small, ordered R domain segment can stabilize CFTR within intracellular compartments, whereas R domain phosphorylation, by interfering with this intracellular stabilization, permits the redistribution of CFTR to the cell surface. Constructs and Antibodies—CFTR deletion constructs were generated via PCR using PFU polymerase (Invitrogen) according to the manufacturer's instructions. PCR-generated deletion constructs were subcloned into pcDNA3.1 using the pcDNA3.1/V5-His TOPO® TA expression kit (Invitrogen) according to instructions. All constructs were sequenced to verify fidelity. Site-directed mutagenesis of residues in the R domain to generate hNEG2-CFTR or sNEG2-CFTR was performed using the GeneTailor™ site-directed mutagenesis system from Invitrogen. For these large modifications, regions were mutated sequentially, with the previously mutated region serving as the template for subsequent mutagenesis reactions. Following the production of each mutation, the entire template was sequenced. Oocyte Preparation—Isolation and cRNA injection of oocytes were performed as described previously (34Cunningham S.A. Worrell R.T. Benos D.J. Frizzell R.A. Am. J. Physiol. 1992; 262: C783-C788Crossref PubMed Google Scholar). In short, Xenopus laevis females were purchased from Xenopus I (Ann Arbor, MI). Surgically isolated oocytes were separated from follicular cells by incubation in 3 mg/ml collagenase (Invitrogen) in calcium-free ND-96 solution at room temperature for 60–90 min followed by devascularization by bathing in 250 mm KH2PO4. Following incubation, stage 5 and 6 oocytes were isolated under a dissecting microscope. The oocytes were allowed to recover overnight in modified Barth's solution (mm) as follows: 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)·4H2O, 0.41 CaCl2·2H2O, 10 HEPES, ½ sodium salt, pH 7.2, with 50 ml of horse serum (Invitrogen) and 0.273 g of sodium pyruvate (Sigma) per liter. The next day, oocytes were injected with 0.5–10 ng of CFTR cRNA (see figure legends). Approximately 25 oocytes were injected with cRNA for each experimental condition, and they were maintained in MBS at 18 °C for 2–3 days prior to current recordings. cRNA was generated using the mMessage mMachine kit (Ambion) for in vitro translation of linearized plasmids. Oocytes were co-injected with cRNA encoding the β2-adrenergic receptor, and stimulation was evoked by addition of 10 μm isoproterenol to the bath. Electrophysiology—Two electrode voltage clamp recordings employed ND-96 as the bath solution (mm) as follows: 96 NaCl, 1 KCl, 1.2 CaCl2, 5.8 MgCl2, 10 HEPES, pH 7.2. Oocytes were impaled with two glass electrodes filled with 3 m KCl; their resistances were 0.2–1.0 megohms. The electrodes were connected to a GeneClamp 500 current/voltage clamp amplifier (Molecular Devices) via Ag-AgCl pellet electrodes and referenced to Ag-AgCl pellet electrodes in the bath. The oocytes were impaled, and the membrane potentials were allowed to stabilize for ∼5 min. The voltage clamp was controlled by an AD/DA interface (AXOLAB 1100), and waveforms were imposed using PC-based software generated in the laboratory. Membrane capacitance (Cm) was calculated on line from the following relation: Cm = τ ((1/Ra) + Gm), where Ra is the access resistance between the current electrode and the oocyte; Gm is the membrane conductance, and τ is the time constant. A 10-mV hyperpolarizing voltage pulse (50 ms duration) was applied to the holding potential of -30 mV to determine Ra, Gm, and τ. As illustrated in Fig. 1A, τ was obtained by fitting the exponential current decay curve during the voltage pulse; in this interval, the current sampling rate was 170 kHz and permitted the acquisition of 1700 data points in 10 ms. Ra was calculated from Vp/Ipp, where Ipp is the instantaneous current obtained from extrapolation of the experimental fit to zero time. The peak of current was detected, and 250 points following the peak were fit using a first-order exponential decay function, indicated as the red overlay in Fig. 1. The steady-state current, Iss, was used to calculate Gm from the average of three points taken near the end of the pulse, using the relation (Iss/(Vp - Ra × Iss)). This approach provides a reliable estimate of Cm both before and after the rather large change in membrane conductance associated with cAMP/PKA stimulation in CFTR expressing oocytes. In the repetitive pulse protocol, Vm was held at -30 mV, approximating the chloride equilibrium potential, and pulsed to -40 mV (50 ms) to obtain Cm, as described. Vm was returned to -30 mV (100 ms) and then pulsed to -60 mV (200 ms) to obtain the reported transmembrane current, Im, which was sampled 10 ms before the end of each pulse. This protocol was repeated every 5 s throughout the experiment. Co-injection of NEG2 Peptides—The NEG2 peptide, derived from the C terminus of the R domain, and two modified peptides (sNEG2 and hNEG2), see figure legends for definition, were synthesized via solid-phase peptide synthesis, kindly provided by Dr. Robert Bridges (Rosalind Franklin University). Each peptide was solubilized at a concentration of 50 μm in an intracellular buffer (35Lang J. Fukuda M. Zhang H. Mikoshiba K. Wollheim C.B. EMBO J. 1997; 16: 5837-5846Crossref PubMed Scopus (101) Google Scholar) containing (mm) the following: 128 potassium glutamate, 5 NaCl, 7 MgSO4, 20 HEPES, pH 7.0, titrated with ultrapure KOH, aliquoted, and stored at -20 °C. Oocytes expressing ΔNEG2-CFTR were voltage-clamped as described above and allowed to equilibrate for 5 min. NEG2, or a modified peptide, was drawn into an injection tip similar to that used for cRNA injections; the clamp was interrupted, and oocytes were impaled and injected with 23 nl of NEG2 peptide solution. Twenty minutes following peptide injection, experimental recordings were initiated using the standard agonist addition and recording protocols described above. Immunoblots and Cell Surface Labeling—Western blotting of Xenopus oocyte extracts was performed as described (36Goldin A.L. Methods Cell Biol. 1991; 36: 487-510Crossref PubMed Scopus (76) Google Scholar). Briefly, oocytes were homogenized in 15 mm Tris, pH 6.8, in 20 μl of buffer/oocyte. An equal volume of 1,1,2-trichloro-trifluorethane (Freon) was added, and the oocytes were spun for 10 min at maximum speed in a bench top centrifuge. The upper phase was recovered, and the remainder was treated again with Freon. Proteins were precipitated with ice-cold methanol and chloroform. Sample buffer was added; the proteins were separated by SDS-PAGE and transferred to Immobilon™-P. Blots were probed with R domain-specific α-hCFTR mouse monoclonal IgG (1:1000) from R & D Systems (Minneapolis, MN) or the mouse monoclonal antibody (1:2500) whose epitope lies at the CFTR C terminus (clone 24-1), which was purified from hybridoma supernatant (HB-11947; ATCC) by Mark Silvis in the laboratory. The secondary antibody was donkey α-mouse horseradish peroxidase (HRP) from Amersham Biosciences (1:5000). Labeling of CFTR at the cell surface utilized an externally epitope-tagged CFTR construct (EXT-CFTR (37Gentzsch M. Chang X.B. Cui L. Wu Y. Ozols V.V. Choudhury A. Pagano R.E. Riordan J.R. Mol. Biol. Cell. 2004; 15: 2684-2696Crossref PubMed Scopus (175) Google Scholar)), kindly provided by Dr. John Riordan (University of North Carolina, Chapel Hill). HEK 293 cells were transiently transfected with cDNA constructs encoding EXT-CFTR or WT CFTR (control), 4 μg/35-mm dish, 24 h prior to experiments using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Following transfection, cells were incubated in HEK 293 media (Dulbecco's modified Eagle's medium + 10% fetal bovine serum) at 37 °C overnight. Cells were stimulated with 10 mm forskolin (EMD Biosciences) at 37 °C for 12 min and placed on ice. For oocyte studies, EXT-CFTR (10 ng) or WT CFTR (1 ng) cRNA was injected together with β-adrenergic receptor and incubated in MBS++ for 3 days at 18 °C. Oocytes were stimulated with 10 μm isoproterenol (Sigma) as in the electrophysiological studies. Nonstimulated HEK cells or oocytes, treated identically except for agonist additions, served as controls. For chemiluminescence measurements, cell surface EXT-CFTR was labeled by sequential incubations in primary monoclonal HA antibody (1:1000, 90 min) (Covance, New York), secondary biotin-conjugated goat anti-mouse IgG (1:200, 90 min) (Invitrogen), and streptavidin conjugated to HRP (1:500; 90 min) (Zymed Laboratories Inc.). All steps were performed at 4 °C to block CFTR trafficking. The cells were then washed extensively, and HRP-labeled proteins were detected using SuperSignal West Femto chemiluminescent substrate (Pierce) and read in a TD20/20 luminometer (Turner, Sunnyvale, CA). Statistics—All data are presented as means ± S.E., where N indicates the number of experiments, and n is the total number of oocytes studied. Statistical analysis was performed using the Student's unpaired t test. A value of p ≤ 0.05 is considered statistically significant, as indicated (by *) in all figures. All experiments were performed on oocytes harvested from at least three X. laevis to judge reproducibility. The functional half-life of different CFTR constructs was determined by fitting current decay curves obtained in the presence of brefeldin A with the two-parameter, single exponential decay regression function of SigmaPlot 2001 (SPSS, Chicago). Calculations—We calculated the number of vesicles (Nv) inserted into the plasma membrane in response to cAMP/PKA stimulation as described by Bertrand et al. (1Bertrand C.A. Frizzell R.A. Am. J. Physiol. 2003; 285: C1-C18Crossref PubMed Scopus (114) Google Scholar), using the equation Nv =ΔCm/Cs·4πrG2, where ΔCm is the measured increase in membrane capacitance; Cs is the specific capacitance of biological membranes (1 μF/cm2), and rG is the radius of a spherical vesicle, assumed to be 100 nm for this purpose. β-Agonist-induced Current and Capacitance Responses—Representative Im and Cm responses from oocytes expressing WT CFTR are illustrated in Fig. 1, A and B, and mean data for basal and peak stimulation values from 15 recordings are summarized in Fig. 1C. In prior experiments of this type, stimulation was produced by addition of forskolin plus IBMX (24Takahashi A. Watkins S.C. Howard M. Frizzell R.A. Am. J. Physiol. 1996; 271: C1887-C1894Crossref PubMed Google Scholar, 25Peters K.W. Qi J. Watkins S.C. Frizzell R.A. Am. J. Physiol. 1999; 277: C174-C180Crossref PubMed Google Scholar). In this study, co-expression of the β2-adrenergic receptor allowed cAMP/PKA stimulation by addition of isoproterenol (10 μm) as employed in previous oocyte studies (38Liu X. Smith S.S. Sun F. Dawson D.C. J. Gen. Physiol. 2001; 118: 433-446Crossref PubMed Scopus (16) Google Scholar, 39Uezono Y. Bradley J. Min C. McCarty N.A. Quick M. Riordan J.R. Chavkin C. Zinn K. Lester H.A. Davidson N. Receptors Channels. 1993; 1: 233-241PubMed Google Scholar). This approach obviates any contribution from the direct interaction of IBMX with CFTR (40Schultz B.D. Frizzell R.A. Bridges R.J. J. Membr. Biol. 1999; 170: 51-66Crossref PubMed Scopus (51) Google Scholar) and was used throughout. Prior to stimulation of oocytes co-expressing CFTR and β2-adrenergic receptor, basal chloride current was 0.10 ± 0.01 μA, and isoproterenol addition elicited a current increase (ΔIm) that averaged 2.5 ± 0.13 μA). This current stimulation was paralleled by a 24% increase in membrane capacitance, ΔCm = 46 ± 9.4 nF, from 192 to 238 nF. These changes in Im and Cm are CFTR-dependent; they are not observed in uninjected oocytes (not shown) or in oocytes expressing β2-adrenergic receptor alone (Fig. 3C). The magnitudes of these responses are consistent with previously published data obtained using forskolin plus IBMX as the cAMP/PKA agonists (1Bertrand C.A. Frizzell R.A. Am. J. Physiol. 2003; 285: C1-C18Crossref PubMed Scopus (114) Google Scholar, 24Takahashi A. Watkins S.C. Howard M. Frizzell R.A
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