CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

Cormet‐Boyaka, Estelle; Di, Anke; Chang, Steven Y.; Naren, Anjaparavanda P.; Tousson, Albert; Nelson, Deborah J.; Kirk, Kevin L.

doi:10.1073/pnas.192203899

Cited by 74 publications

(59 citation statements)

References 37 publications

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“…However, in several respects, the inhibitory mechanism of Shank2 on CFTR is different from that of syntaxin 1A. For example, syntaxin 1A binds to the N-terminal part of CFTR and is believed to regulate CFTR activity by directly modulating the gating properties or by affecting membrane trafficking of CFTR (29). On the contrary, Shank2 seems to bind to the C-terminal PDZ-binding motif of CFTR (Table I and Fig.…”

Section: Discussionmentioning

confidence: 99%

Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Anion-transporting Activities by Shank2

Kim

Han

Namkung

et al. 2004

Journal of Biological Chemistry

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Accumulating evidence suggests that protein-protein interactions play an important role in transepithelial ion transport. In the present study, we report on the biochemical and functional association between cystic fibrosis transmembrane conductance regulator (CFTR) and a PDZ domain-containing protein Shank2. Exploratory reverse transcription-PCR screening revealed mRNAs for several members of PDZ domain-containing proteins in epithelial cells. Shank2, one of these scaffolding proteins, showed a strong interaction with CFTR by yeast two-hybrid assays. Shank2-CFTR interaction was verified by co-immunoprecipitation experiments in mammalian cells. Notably, this interaction was abolished by mutations in the PDZ domain of Shank2. Protein phosphorylation, HCO 3 ؊ transport and Cl ؊ current by CFTR were measured in NIH 3T3 cells with heterologous expression of Shank2. Of interest, expression of Shank2 suppressed cAMP-induced phosphorylation and activation of CFTR. Importantly, loss of Shank2 by stable transfection of antisense-hShank2 plasmid strongly increased CFTR currents in colonic T84 cells, in which CFTR and Shank2 were natively expressed. Our results indicate that Shank2 negatively regulates CFTR and that this may play a significant role in maintaining epithelial homeostasis under normal and diseased conditions such as those presented by secretory diarrhea.Secretory epithelia perform vectorial transport of salt and water molecules by coordinated actions of the transporters expressed in polarized epithelial membranes. One of the key membrane proteins regulating overall fluid movements is the cystic fibrosis transmembrane conductance regulator (CFTR), 1 which itself has an anion-transporting activity (1-3). Aberrant membrane transport caused by either hypo-or hyper-functioning of CFTR, can be detrimental, and may result in life-threatening diseases, such as cystic fibrosis or secretory diarrhea (4, 5). Therefore, the fine regulation of salt and water transport is essential in epithelial and body homeostasis.Accumulating evidence suggests that protein-protein interaction performs an important role in the regulation of transepithelial ion transport (6). Clustering of ion transporters and associated proteins in microdomains of polarized epithelia can facilitate the effective secretion or absorption of salt and water molecules. In this regard, modular adaptor proteins such as PDZ (PSD-95/discs large/ZO-1) domain-containing proteins have drawn increasing attention due to their ability to form supramolecular complexes (7). We have previously shown that the regulatory interaction between CFTR and Na ϩ /H ϩ exchanger 3 (NHE3) through PDZ-based scaffolds is essential for the coordinated regulation of pancreatic bicarbonate secretion (8). In addition, it was found that a number of membrane transporters and receptors participating in pancreatic fluid formation, such as Na ϩ -HCO 3 Ϫ cotransporters (NBC), purinergic receptors, and secretin receptors have a PDZ-binding motif on their C terminus (9, 10). Therefore, multiple pro...

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Section: Discussionmentioning

confidence: 99%

Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Anion-transporting Activities by Shank2

Kim

Han

Namkung

et al. 2004

Journal of Biological Chemistry

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“…An affinity-purified polyclonal antibody raised against human SNAP-23 and a mouse monoclonal antibody raised against the COOH-terminal tail of human CFTR have been described earlier (15). A mouse monoclonal antibody raised against Munc-18a was purchased from Transduction Laboratories.…”

Section: Methodsmentioning

confidence: 99%

The Interaction between Syntaxin 1A and Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Is Mechanistically Distinct from Syntaxin 1A-SNARE Interactions

Ganeshan¹,

Di²,

Nelson³

et al. 2003

Journal of Biological Chemistry

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Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channels and synaptic Ca 2؉ channels in addition to participating in SNARE complex assembly and membrane fusion. We exploited the isoform-specific nature of the interaction between syntaxin 1A and CFTR to identify residues in the H3 domain of this SNARE (SNARE motif) that influence CFTR binding and regulation. Mutating isoform-specific residues that map to the surface of syntaxin 1A in the SNARE complex led to the identification of two sets of hydrophilic residues that are important for binding to and regulating CFTR channels or for binding to the syntaxin regulatory protein Munc-18a. None of these mutations affected syntaxin 1A binding to other SNAREs or the assembly and stability of SNARE complexes in vitro. Conversely, the syntaxin 1A-CFTR interaction was unaffected by mutating hydrophobic residues in the H3 domain that influence SNARE complex stability and Ca 2؉ channel regulation. Thus, CFTR channel regulation by syntaxin 1A involves hydrophilic interactions that are mechanistically distinct from the hydrophobic interactions that mediate SNARE complex formation and Ca 2؉ channel regulation by this t-SNARE.

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“…SNAP23 physically interacts with CFTR by binding to its amino-terminal tail and inhibits CFTR chloride currents by influencing channel gating. 23 Using the Human Splicing Finder tool, we found a change in a potential branch point. The CV for the sequence with the C allele (0-100) is 80.91, whereas the CV for the sequence with the T allele is 57.07 leading to an abolished splice site.…”

Section: Discussionmentioning

confidence: 98%

“…The genes interacting with CFTR are SNAP23 (synaptosomal-associated protein, 23kDa (Annexin A5) and PPP2R4, PPP2R1A, PPP2R5E, that encode three regulatory subunits of protein phosphatase 2A. [23][24][25][26][27][28][29] The two genes interacting with EnaC are Nedd4L (neural precursor cell expressed, developmentally downregulated 4-like) and PRSS8 (prostasin). [30][31][32] The last group consists of three genes that are involved in CFTR trafficking, AHSAI (activator of heat shock 90-kDa protein ATPase homolog 1), CALR (calreticulin) and KRT19 (cytokeratin 19).…”

Section: Introductionmentioning

confidence: 99%

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

et al. 2012

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There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (Pr0.01), the specific effective airway resistance (Pr0.005) and the forced expiratory volume in 1 s (Pr0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.

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CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

Cited by 74 publications

References 37 publications

Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Anion-transporting Activities by Shank2

Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Anion-transporting Activities by Shank2

The Interaction between Syntaxin 1A and Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Is Mechanistically Distinct from Syntaxin 1A-SNARE Interactions

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

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