Up-regulation of Acid-gated Na+ Channels (ASICs) by Cystic Fibrosis Transmembrane Conductance Regulator Co-expression in Xenopus Oocytes

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Proton-gated Na؉ channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na ؉ currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na ؉ current in Calu-3 cells from ؊154 ؎ 28 to ؊33 ؎ 16 pA (n ‫؍‬ 5, p < 0.05) at ؊100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system.

“…5D). These results were consistent with our previous observation that elevation of cAMP does not have an effect on ␣␤␥ ENaC and ASIC1a/2a channels expressed in oocytes (8,39).…”

Section: Journal Of Biological Chemistry 36963supporting

confidence: 82%

Section: Asic3 Interacts With Cftrcontrasting

confidence: 52%

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Interregulation of Proton-gated Na+ Channel 3 and Cystic Fibrosis Transmembrane Conductance Regulator

Shrestha

et al. 2006

Self Cite

“…Finally, ASIC function can also be regulated via association with the cystic fibrosis transmembrane conductance regulator, both a Cl Ϫ channel and an epithelial transport regulator. This channel can interact with the heteromultimeric ASIC1a-ASIC2a complex and increase the Na ϩ current passing through the pore of the complex (38). Here, we have shown that p11 increases ASIC1a expression at the cell surface.…”

Section: Discussionmentioning

confidence: 55%

Annexin II Light Chain p11 Promotes Functional Expression of Acid-sensing Ion Channel ASIC1a

Donier

Rugiero

Okuse

et al. 2005

Acid-sensing ion channels (ASICs) have been implicated in a wide variety of physiological functions. We have used a rat dorsal root ganglion cDNA library in a yeast two-hybrid assay to identify sensory neuron proteins that interact with ASICs. We found that annexin II light chain p11 physically interacts with the N terminus of ASIC1a, but not other ASIC isoforms. Immunoprecipitation studies confirmed an interaction between p11 and ASIC1 in rat dorsal root ganglion neurons in vivo. Coexpression of p11 and ASIC1a in CHO-K1 cells led to a 2-fold increase in expression of the ion channel at the cell membrane as determined by membrane-associated immunoreactivity and cell-surface biotinylation. Consistent with these findings, peak ASIC1a currents in transfected CHO-K1 cells were up-regulated 2-fold in the presence of p11, whereas ASIC3-mediated currents were unaffected by p11 expression. Neither the pH dependence of activation nor the rates of desensitization were altered by p11, suggesting that its primary role in regulating ASIC1a activity is to enhance cell-surface expression of ASIC1a. These data demonstrate that p11, already known to traffic members of the voltage-gated sodium and potassium channel families as well as transient receptor potential and chloride channels, also plays a selective role in enhancing ASIC1a functional expression.

“…Unlike PICK1 (30, 31) for ASIC2a, cotransfection with CIPP increases the ASIC3 current membrane density and produces a shift in the pH-dependent activation. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been shown to regulate ASICs; however, the physiological relevance of this regulation is not known (40).…”

Section: Fig 2 Effect Of Pick1 On Asic2a Ph Dependence and Current mentioning

confidence: 99%

Protein Kinase C Stimulates the Acid-sensing Ion Channel ASIC2a via the PDZ Domain-containing Protein PICK1

Baron¹,

Deval²,

Salinas

et al. 2002

110

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in central and sensory neurons where they are involved in neuromodulation and in pain perception. Recently, the PDZ domain-containing protein PICK1 (protein interacting with C-kinase) has been shown to interact with ASIC1a and ASIC2a, raising the possibility that protein kinase C (PKC) could regulate ASICs. We now show that the amplitude of the ASIC2a current, which was only modestly increased (ϳ؉30%) by the PKC activator 1-oleyl-2-acetyl-sn-glycerol (OAG, 50 M) in the absence of PICK1, was strongly potentiated (ϳ؉300%) in the presence of PICK1. This PICK1-dependent regulatory effect was inhibited in the presence of a PKC inhibitory peptide and required the PDZ domain of PICK1 as well as the PDZ-binding domain of ASIC2a. We have also shown the direct PICK1-dependent phosphorylation of ASIC2a by [ 32 P]phosphate labeling and immunoprecipitation and identified a major phosphorylation site, 39 TIR, on the N terminus part of ASIC2a. The OAG-induced increase in ASIC2a current amplitude did not involve any change in the unitary conductance of the ASIC2a channel, whether co-expressed with PICK1 or not. These data provide the first demonstration of a regulation of ASICs by protein kinase phosphorylation and its potentiation by the partner protein PICK1.