New paradigms of CFTR chloride channel regulation
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Chloride channel
Epithelial sodium channel
Epithelial sodium channel
Chloride channel
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Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.
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The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.
Chloride channel
Epithelial sodium channel
Transepithelial potential difference
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Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by protein kinase A (PKA) phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing one to three protein segments of the R domain including residues 708-759 (ΔR708-759), R760-783, and R784-835, each of which contains one or two PKA phosphorylation sites. Deletion of R708-759 or R760-783 had little effect on CFTR gating, whereas all mutations lacking R784-835 reduced CFTR activity by decreasing the mean burst duration and increasing the interburst interval (IBI). The data suggest that R784-835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ΔR784-835 had minor impact on gating potentiation by 2'dATP, CaATP, and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R809-835 shortened the IBI of ΔR708-835-CFTR. Consistently, ΔR815-835, but not ΔR784-814, enhanced IBI, whereas both reduced mean burst duration. These data suggest that the entirety of R784-835 is required for stabilizing the open state of CFTR; however, R815-835, through interactions with the channel, is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR708-783-CFTR. Conversely, the IBI of ΔR708-814-CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R784-814-mediated autoinhibition that prevents IBI shortening by R815-835 This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.
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The cystic fibrosis transmembrane conductance regulator (CFTR), which forms adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, is defective in patients with cystic fibrosis. This protein contains two putative nucleotide binding domains (NBD1 and NBD2) and an R domain. CFTR in which the R domain was deleted (CFTR delta R) conducted chloride independently of the presence of cAMP. However, sites within CFTR other than those deleted also respond to cAMP, because the chloride current of CFTR delta R increased further in response to cAMP stimulation. In addition, deletion of the R domain suppressed the inactivating effect of a mutation in NBD2 (but not NBD1), a result which suggests that NBD2 interacts with the channel through the R domain.
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Cyclic nucleotide-binding domain
Cyclic adenosine monophosphate
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A Synthetic Chloride Channel Restores Chloride Conductance in Human Cystic Fibrosis Epithelial Cells
Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl−) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl− transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl− channels to mediate Cl− transport across lipid bilayer membranes is capable of restoring Cl− permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl− channel dysfunction.
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SUMMARY 1. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the primary defect observed in patients with cystic fibrosis. 2. The CFTR is a member of the ATPase‐binding cassette (ABC) transporter family but, unlike other members of this group, CFTR conducts a chloride current that is activated by cAMP. 3. In epithelial cells, the cAMP‐stimulated chloride current is conducted by both CFTR and the outwardly rectifying chloride channel (ORCC). 4. The present review summarizes the current knowledge of the properties of the two channels, as well as their relationship. Because the gene encoding the ORCC has not been identified, a discussion as to possible candidates for this chloride channel is included.
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Chloride channel
Vas deferens
Loss function
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Chloride channel
Epithelial sodium channel
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Chloride channels are widely expressed and play important roles in cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. Most chloride channels have yet to be identified at a molecular level. The ClC gene family and the cystic fibrosis transmembrane conductance regulator (CFTR) are distinct chloride channels expressed in many cell types, and mutations in their genes are the cause of several diseases including myotonias, cystic fibrosis, and kidney stones. Because of their molecular definition and roles in disease, these channels have been studied intensively over the past several years. The focus of this review is on recent studies that have provided new insights into the mechanisms governing the opening and closing, i.e. gating, of the ClC and CFTR chloride channels.
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