Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Gen Physiol
2001 Oct 01;1184:433-46. doi: 10.1085/jgp.118.4.433.
Show Gene links
Show Anatomy links
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.
Liu X
,
Smith SS
,
Sun F
,
Dawson DC
.
???displayArticle.abstract???
Some studies of CFTR imply that channel activation can be explained by an increase in open probability (P(o)), whereas others suggest that activation involves an increase in the number of CFTR channels (N) in the plasma membrane. Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+). Covalent modification of R334C CFTR with MTSET+ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Thus, labeled and unlabeled channels could be differentiated by noting the percent decrease in conductance brought about by exposure to 2-ME. When oocytes were briefly (20 s) exposed to MTSET+ before CFTR activation, the subsequently activated conductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSET+. The addition of unlabeled, newly synthesized channels to the plasma membrane could be monitored on-line during the time when the rate of addition was most rapid after cRNA injection. The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of approximately 24-48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. These findings demonstrate that in Xenopus oocytes, the major mechanism of CFTR activation by cAMP is by means of an increase in the open probability of CFTR channels.
???displayArticle.pubmedLink???
11585853
???displayArticle.pmcLink???PMC2233697 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. I-V relationship of R334C CFTR is modified by MTSET+ and MTSES−. (A) I-V plots at steady-state activation. Oocytes were continuously perfused with a cocktail containing 10 μM isoproterenol and 1 mM IBMX (control). An ∼5-min exposure to 1 mM MTSET+ induced an approximate doubling of the conductance and a change in the shape of the I-V plot. (B) I-V plots obtained at steady-state activation (control) and after ∼5-min exposure to 1 mM MTSES− that attenuated the conductance by ∼50% and enhanced inward rectification.
Figure 2. Modification of R334C CFTR by MTSET+ was stable for at least 5 h. The conductance (gCl@Erev) was first obtained 30–40 min after the initial exposure of the oocytes to stimulatory cocktail when the activation of the Cl− conductance had attained a steady state (control). Each oocyte was exposed to 100 μM or 1 mM MTSET+ for 20 s. The MTSET+-containing stimulatory cocktail was then replaced with stimulatory cocktail lacking the thiol reagent, and the oocytes were continuously perfused for up to 5 h. The conductances measured at 2 min, 2 h, and 5 h after exposure of oocytes to MTSET+ were normalized to the steady-state conductance before exposure to MTSET+.
Figure 3. The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with MTSET+ before the activation. Records of gCl versus time obtained from R334C CFTR expressing oocytes that were obtained from the same frog and assayed on the same day. Oocytes were always perfused with frog Ringer's unless noted (see materials and methods). After a control period, they were perfused with stimulatory cocktail containing 10 μM isoproterenol and 1 mM IBMX (Isop + IBMX). (A) A record of gCl versus time showing that at the steady-state activation, MTSET+ caused about a doubling of gCl. (B) A record of gCl versus time showing that the activated gCl at the steady state of an oocyte preexposed to MTSET+ was much higher than the gCl at unmodified condition as seen in A, and 2-ME reduced gCl to ∼50% of the maximum gCl. (C) A record of gCl versus time of showing that the exposure to MTSES− after prelabeling with MTSET+ had no effect on gCl before and after activation.
Figure 4. MTSET+ labeling did not affect the activation and inactivation process of R334C CFTR. A record of the conductance measured at the reversal potential throughout an experiment. After activation, an oocyte was exposed to 100 μM MTSET+, and then was inactivated without removing MTSET+. The oocyte was then reactivated, showing a gCl similar to the gCl before inactivation. The gCl was then reduced to ∼50% by 2-ME.
Figure 5. The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with 20-s exposure to MTSET+ before the activation. (A) A record of the conductance measured at the reversal potential throughout an experiment. (B) Exposure to 1 mM MTSET+ or 1 mM MTSES− had no significant effects on the background conductance before activation of CFTR (1 and 2). (C) Modification by 1 mM MTSET+ for 20 s before activation prevented any further modification by MTSES+ or MTSET+ during or after activation (3–5), and 2-ME reversed the effect of MTSET+ (6). (D) Further modification by MTSET+ was possible after 2-ME treatment and was also reversed by 2-ME (7 and 8). The apparent lower values of Erev (∼17 mV) resulted from a shift in the tip potential of the recording electrode.
Figure 6. Time-dependent increase in the conductance of R334C CFTR in the first 5 d after cRNA injection. Oocyte conductance in both inactive and cAMP-activated states was measured on days 1, 2, 3, 4, and 5 (24, 48, 72, 96, and 120 h) after cRNA injection and the values were normalized to the conductance on day 2.
Figure 7. The addition of new channels can be monitored by labeling preexisting channels with MTSET+. (A) A record of the conductance measured at the reversal potential throughout an experiment on day 1. The conductance was increased after 4.5 h due to the addition of new, unlabeled channels. (B) A record of the conductance measured at the reversal potential throughout an experiment on day 5. No increase in the conductance was observed during the 4.5-h period.
Figure 8. BFA prevented the addition of new channels on day 1, and had no effect on the conductance on day 5. Records of the conductance measured at the reversal potential throughout experiments. Oocytes were exposed to BFA throughout the entire experimental period. No increase in the conductance with time was observed after 4.5 h on either day 1 (A) or day 5 (B).
Figure 9. Long-term BFA treatment prevented the addition of new channels. Some oocytes were kept in MBSH solution as usual (control). Some oocytes were transferred to the MBSH solution containing 5 μM BFA a few hours after cRNA injection and remained in the same solution thereafter (BFA-treated). The background conductance (gbkg) and cAMP-elevated Cl− conductance (gCl) were assayed at 24 and 48 h after cRNA injection.
Anderson,
Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.
1991, Pubmed
Anderson,
Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.
1991,
Pubmed
Anderson,
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
1992,
Pubmed
Anderson,
Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains.
1992,
Pubmed
Anthony,
Molecular characterization of antipeptide antibodies against the 5-HT1A receptor: evidence for state-dependent antibody binding.
1997,
Pubmed
Aoki,
Beta-adrenergic receptors: astrocytic localization in the adult visual cortex and their relation to catecholamine axon terminals as revealed by electron microscopic immunocytochemistry.
1992,
Pubmed
Baukrowitz,
Coupling of CFTR Cl- channel gating to an ATP hydrolysis cycle.
1994,
Pubmed
Bear,
Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene.
1991,
Pubmed
,
Xenbase
Carson,
The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity.
1995,
Pubmed
Chen,
The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane.
2001,
Pubmed
Cheng,
Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.
1991,
Pubmed
Denning,
Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.
1992,
Pubmed
Dho,
Plasma membrane recycling in CFTR-expressing CHO cells.
1993,
Pubmed
Drumm,
Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes.
1991,
Pubmed
,
Xenbase
Fischer,
CFTR displays voltage dependence and two gating modes during stimulation.
1994,
Pubmed
Gadsby,
Regulation of CFTR channel gating.
1994,
Pubmed
Holmgren,
On the use of thiol-modifying agents to determine channel topology.
1996,
Pubmed
,
Xenbase
Howard,
cAMP-regulated trafficking of epitope-tagged CFTR.
1996,
Pubmed
,
Xenbase
Howard,
Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells.
2000,
Pubmed
Hug,
The role of exocytosis in the activation of the chloride conductance in Chinese hamster ovary cells (CHO) stably expressing CFTR.
1997,
Pubmed
Hwang,
Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis.
1994,
Pubmed
Javitch,
Constitutive activation of the beta2 adrenergic receptor alters the orientation of its sixth membrane-spanning segment.
1997,
Pubmed
Kartner,
Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance.
1991,
Pubmed
Klausner,
Brefeldin A: insights into the control of membrane traffic and organelle structure.
1992,
Pubmed
Lehrich,
Vasoactive intestinal peptide, forskolin, and genistein increase apical CFTR trafficking in the rectal gland of the spiny dogfish, Squalus acanthias. Acute regulation of CFTR trafficking in an intact epithelium.
1998,
Pubmed
,
Xenbase
Loffing,
Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells.
1998,
Pubmed
Lukacs,
Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation.
1997,
Pubmed
Mansoura,
Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore.
1998,
Pubmed
,
Xenbase
Moyer,
Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells.
1998,
Pubmed
Peacock,
Human low density lipoprotein receptor expressed in Xenopus oocytes. Conserved signals for O-linked glycosylation and receptor-mediated endocytosis.
1988,
Pubmed
,
Xenbase
Peters,
Syntaxin 1A inhibits regulated CFTR trafficking in xenopus oocytes.
1999,
Pubmed
,
Xenbase
Prince,
Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal.
1999,
Pubmed
Prince,
Cell surface labeling of CFTR in T84 cells.
1993,
Pubmed
Prince,
Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.
1994,
Pubmed
Quinton,
Physiological basis of cystic fibrosis: a historical perspective.
1999,
Pubmed
Rich,
Effect of deleting the R domain on CFTR-generated chloride channels.
1991,
Pubmed
Santos,
Activation of the cystic fibrosis transmembrane regulator by cyclic AMP is not correlated with inhibition of endocytosis.
1994,
Pubmed
Schwiebert,
Heterotrimeric G proteins, vesicle trafficking, and CFTR Cl- channels.
1994,
Pubmed
Sheppard,
Structure and function of the CFTR chloride channel.
1999,
Pubmed
Smit,
Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
1993,
Pubmed
,
Xenbase
Smith,
CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.
2001,
Pubmed
,
Xenbase
Smith,
Expression of rat mRNA coding for hormone-stimulated adenylate cyclase in Xenopus oocytes.
1987,
Pubmed
,
Xenbase
Snyder,
Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na(+) channel to the cell surface.
2000,
Pubmed
Sood,
Regulation of CFTR expression and function during differentiation of intestinal epithelial cells.
1992,
Pubmed
Tabcharani,
Phosphorylation-regulated Cl- channel in CHO cells stably expressing the cystic fibrosis gene.
1991,
Pubmed
Takahashi,
CFTR-dependent membrane insertion is linked to stimulation of the CFTR chloride conductance.
1996,
Pubmed
,
Xenbase
Tousson,
Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments.
1996,
Pubmed
Vassilev,
Identification of an intracellular peptide segment involved in sodium channel inactivation.
1988,
Pubmed
Vassilev,
Inhibition of inactivation of single sodium channels by a site-directed antibody.
1989,
Pubmed
Weber,
Capacitance measurements reveal different pathways for the activation of CFTR.
1999,
Pubmed
,
Xenbase
Wilkinson,
CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.
1996,
Pubmed
,
Xenbase
Wilkinson,
CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain.
1997,
Pubmed
,
Xenbase
Winter,
Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation.
1994,
Pubmed
Yang,
Molecular basis of charge movement in voltage-gated sodium channels.
1996,
Pubmed
Zeltwanger,
Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.
1999,
Pubmed
