Button B , Reuss L , Altenberg GA .

CA-72783 NCI NIH HHS , DK-38734 NIDDK NIH HHS

	Figure 1. Activation of CFTR Cl− conductance by PKA stimulation. The two-electrode voltage-clamp technique was used to measure ionic currents before and ∼30 min after exposure to a cAMP cocktail consisting of 250 μM 8-Br-cAMP and 25 μM forskolin (cAMP). (A) Representative currents obtained in water-injected oocytes and oocytes expressing hCFTR or XCFTR. (B) Time course of hCFTR and XCFTR activation by cAMP. ΔI is the current measured at 0 mV minus the current at −30 mV (holding potential). (C) Halide-selectivity sequence of cAMP-activated hCFTR. I-V relationships were obtained from an oocyte in the constant presence of cAMP cocktail by substituting NaCl in each solution with the corresponding sodium halide salt for 2 min before the I-V plots. Currents were measured at 400 ms after the start of voltage pulses ranging from −100 to 30 mV at 10-mV intervals. (D) Halide-selectivity sequence obtained from a XCFTR-expressing oocyte. Injection of hCFTR and XCFTR cRNAs elicits anion currents with the expected halide selectivity.
	Figure 3. Effects of PMA and forskolin on cAMP levels. Data are from unstimulated oocytes and oocytes exposed for 20 min to either PMA (250 nM) or forskolin (25 μM). Forskolin, but not PMA, increased oocytes cAMP (P < 0.01, n = 3 for each group of oocytes).
	Figure 2. Activation of CFTR Cl− conductance by PKC. (A) Representative I-V plot obtained from an oocyte injected with hCFTR. (B) I-V plot obtained from an oocyte injected with XCFTR. (C) Time course of hCFTR activation by PMA. (D) Time course of XCFTR activation by PMA. (E) Summary of the maximal conductance elicited by PMA. Data were normalized to the maximal conductance obtained with PMA + cAMP. Data shown are from 10 to 12 hCFTR- and XCFTR-expressing oocytes, respectively. I-V plots in PMA-treated oocytes were obtained approximately 15 min after exposure to 250 nM PMA. Next, the cells were exposed to the cAMP cocktail in the continuous presence of PMA, and I-V plots were recorded approximately 10 min later. The results show that PMA is highly effective in activating XCFTR, but not hCFTR. See Fig. 1 for additional details.
	Figure 4. Alignment of the sequences of human, Xenopus and Necturus CFTR R domains. The number below each consensus sequence refers to the Ser/Thr position of the full-length hCFTR sequence. Arrows above indicate that the labeled residue has been shown to be phosphorylated by either PKA or PKC in human CFTR (Cheng et al. 1991; Picciotto et al. 1992).
	Figure 5. Design and expression of the human-Xenopus chimera (HXH-CFTR). (A) Schematic representation of hCFTR, XCFTR, and HXH-CFTR, showing the location of the membrane-spanning domain (MSD), nucleotide-binding domain (NBD), and the R domain. (B) Plasma membrane expression of hCFTR, XCFTR, and HXH-CFTR chimera. Western blots of biotinylated membranes injected with water [C], HXH-CFTR [HXH], hCFTR [H], or XCFTR [X]; there were five oocytes per condition. The arrow denotes the expected size of full-length CFTR (∼170 kD). (C) Representative currents from an oocyte expressing HXH-CFTR. (D) Halide selectivity sequences of cAMP-activated HXH-CFTR. The results show that PKA stimulation elicits a near-linear current with reversal potential near ECl and halide selectivity identical to that of wild-type hCFTR (Br− = Cl− > I− > F−). See Fig. 1 for additional details.
	Figure 6. Stimulation of HXH-CFTR by PKC-mediated phosphorylation. (A) I-V plot from an oocyte expressing HXH-CFTR. (B) I-V plot from an oocyte expressing hCFTR. (C) Time course of HXH-CFTR activation by PMA. Priming denotes the addition of 50 μM 8-Br-cAMP. (D) Comparison of CFTR activation by PMA in oocytes expressing either wild-type hCFTR or XCFTR, or the HXH-CFTR chimera. Data were obtained in oocytes primed with 50 μM cAMP (see text), and were normalized to the current elicited by PMA and cAMP cocktail together. Data shown are from 12, 12, and 15 experiments in hCFTR-, XCFTR-, and HXH-CFTR-expressing oocytes, respectively. The results show that the HXH-CFTR chimera is fully sensitive to PMA stimulation. See Fig. 1 for additional details.
	Figure 7. Knockout of the unique PKC consensus phosphorylation sites prevents full activation of XCFTR by PMA. (A) Representative I-V relationships from an oocyte expressing the double knockout of conserved PKC consensus phosphorylation sites (S686A/S790A-XCFTR). (B) I-V plot from a cell expressing the double knockout of unique PKC consensus phosphorylation sites (T665A/S694A-XCFTR). (C) Summary of results from experiments identical to those in A (n = 4) and B (n = 6). The results indicate that at least one of the conserved PKC consensus sites is necessary for full activation by PMA. See Fig. 1 and Fig. 2 for additional details.
	Figure 8. Thr665 is critical for activation of XCFTR by PKC-mediated phosphorylation. (A) Representative I-V plot from an oocyte expressing a single knockout of the unique PKC consensus phosphorylation site containing Ser694 (S694A-XCFTR). (B) I-V plot from an oocyte expressing a single knockout of the unique PKC consensus phosphorylation site containing Thr665 (Thr665/A-XCFTR). (C) Summary of results from experiments identical to those in A (n = 6) and B (n = 14). The results denote a critical role of Thr665 in the stimulation of XCFTR by PMA. See Fig. 1 and Fig. 2 for additional details.
	Figure 9. The mutation Thr665 to Ala does not increase cAMP-activated XCFTR currents. Time course of the currents after stimulation with the cAMP cocktail in oocytes injected with wild-type XCFTR, S686A/S790A-XCFTR, or T665A/S694A-XCFTR cRNAs. Currents were measured at 30 mV, 20 min after exposure to the cAMP cocktail. The data show that the Thr665 to Ala mutation has no significant effect on the level of cAMP-activated currents.
	Figure 10. PMA produces a large increase in conductance in a mutant hCFTR (H667R-hCFTR) with an engineered PKC consensus phosphorylation sequence that includes Thr665. (A) Representative I-V plot from an oocyte expressing H667R-hCFTR. (B) Summary of results from experiments such as those in A. Oocytes were primed with 25 μM 8-Br-cAMP. Data were obtained from six experiments. The results indicate that the engineered PKC consensus site is sufficient to confer a large response to PKC stimulation.

Anderson, Nucleoside triphosphates are required to open the CFTR chloride channel. 1991, Pubmed

Anderson, Nucleoside triphosphates are required to open the CFTR chloride channel. 1991, Pubmed
Baldursson, Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia. 2000, Pubmed
Bear, Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene. 1991, Pubmed , Xenbase
Berger, Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases. 1993, Pubmed
Chan, Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator's NH(2)-terminal nucleotide binding domain. 2000, Pubmed , Xenbase
Chang, Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. 1993, Pubmed
Cheng, Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. 1991, Pubmed
Cohn, CFTR: development of high- affinity antibodies and localization in sweat gland. 1991, Pubmed
Copello, cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block. 1993, Pubmed
Cunningham, cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA. 1992, Pubmed , Xenbase
Dulhanty, Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator. 1994, Pubmed
Gadsby, Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. 1999, Pubmed
Gao, A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways. 1999, Pubmed
Han, P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody. 1996, Pubmed
Heming, Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium. 1994, Pubmed
Hung, Crystal structure of the ATP-binding subunit of an ABC transporter. 1998, Pubmed
Jia, Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. 1997, Pubmed
Ma, Stimulatory and Inhibitory Functions of the R Domain on CFTR Chloride Channel. 2000, Pubmed
Mo, Comparison of amphibian and human ClC-5: similarity of functional properties and inhibition by external pH. 1999, Pubmed , Xenbase
Nagel, The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator. 1992, Pubmed
Ostedgaard, A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution. 2000, Pubmed
Pajor, Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1. 1998, Pubmed , Xenbase
Picciotto, Phosphorylation of the cystic fibrosis transmembrane conductance regulator. 1992, Pubmed
Price, Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR. 1996, Pubmed , Xenbase
Rich, Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain. 1993, Pubmed
Seibert, Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein. 1997, Pubmed
Sharon, Positive and negative coupling of the metabotropic glutamate receptors to a G protein-activated K+ channel, GIRK, in Xenopus oocytes. 1997, Pubmed , Xenbase
Sheppard, Structure and function of the CFTR chloride channel. 1999, Pubmed
Tabcharani, Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene. 1991, Pubmed
Torres, Polarized expression of cAMP-activated chloride channels in isolated epithelial cells. 1996, Pubmed
Vankeerberghen, Functional characterization of the CFTR R domain using CFTR/MDR1 hybrid and deletion constructs. 1999, Pubmed
Vanoye, P-glycoprotein is not a swelling-activated Cl- channel; possible role as a Cl- channel regulator. 1997, Pubmed
Wilkinson, CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. 1997, Pubmed , Xenbase
Yamazaki, Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C. 1999, Pubmed , Xenbase