Cloning and heterologous expression of new xANO2 from Xenopus laevis

Ryu, Rae Hyung; Oh, Soo Jin; Lee, Ra Mi; Jeong, Seong Won; Jan, Yuh Nung; Lee, Chi H.; Lee, C. Justin; Jeong, Se-Min

doi:10.1016/j.bbrc.2011.04.060

Cited by 4 publications

(5 citation statements)

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“…This cDNA was similar to the rat CLCA sequences showing 56% homology for the nucleotide sequence, and a 50% homology for the amino acid sequence [15,16]. There was a low 21% similarity in the restricted partial region of xANO2 [9]. CMP6 shows the most homology (over 50%) to mice CLCA1 and rbCLCA1 (Table 2).…”

Section: Discussionmentioning

confidence: 71%

“…Recently, two different CaCCs, xTMEM16A and xANO2, were cloned as Xenopus laevis ( X. laevis ) CaCCs [8,9]. These two TMEM16 channels, also called anoctamin, have very significant molecular and physiological characteristics.…”

mentioning

confidence: 99%

“…These two TMEM16 channels, also called anoctamin, have very significant molecular and physiological characteristics. In our research of anoctamin, tissue distribution analysis revealed a ubiquitous expression pattern in various X. laevis tissues including intestines [9]. …”

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confidence: 99%

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Isolation and Expression Profile of the Ca²⁺-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis

et al. 2011

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To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA.

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Section: Discussionmentioning

confidence: 71%

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confidence: 99%

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Isolation and Expression Profile of the Ca²⁺-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis

et al. 2011

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“…tropicalis tmem16a is expressed in mucin-producing small secretory cells in tadpole skin Functional studies of Tmem16a in Xenopus are limited to its role in the oocyte and the prevention of polyspermy [39,40,41]. RT-PCR expression analysis in adults shows expression in various tissues [42,43], while bulk RNA sequencing in developing embryos shows expression at Nieuwkoop-Faber (NF) stages NF1, NF9 and NF24-NF42 [42]. We therefore sought tissue-speci c expression during skin development.…”

Section: Resultsmentioning

confidence: 99%

The Tmem16a chloride channel is required for mucin maturation after secretion from goblet-like cells in the Xenopus tropicalis tadpole skin.

Dubaissi,

Emma,

Lilley

et al. 2024

Preprint

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The TMEM16A chloride channel is proposed as a therapeutic target in cystic fibrosis, where activation of this ion channel might restore airway surface hydration and mitigate respiratory symptoms. While TMEM16A is associated with increased mucin production under stimulated or pro-inflammatory conditions, its role in baseline mucin production, secretion and/or maturation is less well understood. Here, we use the Xenopus tadpole skin mucociliary surface as a model of human upper airway epithelium to study Tmem16a function in mucus production. We found that Xenopus tropicalis Tmem16a is present at the apical membrane surface of tadpole skin small secretory cells that express canonical markers of mammalian “goblet cells” such as Foxa1 and Spdef. X. tropicalis Tmem16a functions as a voltage-gated, calcium-activated chloride channel when transfected into mammalian cells in culture. Depletion of Tmem16a from the tadpole skin results in dysregulated mucin maturation post-secretion, with secreted mucins having a disrupted molecular size distribution and altered morphology assessed by sucrose gradient centrifugation and electron microscopy, respectively. Our results show that in the Xenopus tadpole skin Tmem16a is necessary for normal mucus barrier formation and demonstrate the utility of this model system to discover new biology relevant to human mucosal biology in health and disease.

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“…In summary, this result indicates a positive modulation of the CaCCs by 1 μM of GSK1016790A in Xenopus oocytes. As mentioned above TMEM16A and TMEM16B are the molecular components that give rise to the CaCCs in Xenopus oocytes [ 50 ]. To explore whether GSKa targets a TMEM16 ion channels, we evaluated its effect on the cloned mouse TMEM16B mediated currents expressed in HEK293 cells.…”

Section: Resultsmentioning

confidence: 99%

Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

Hernández¹,

Alaniz-Palacios²,

Contreras-Vite³

et al. 2021

Biochemistry and Biophysics Reports

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Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes and their malfunction is implicated in diverse pathologies such as cancer, asthma, and hypertension. TMEM16A and TMEM16B proteins are the structural components of the CaCCs. Recent studies in cell cultures and animal models have demonstrated that pharmacological inhibition of CaCCs could be helpful in the treatment of some diseases, however, there are few specific modulators of these channels. CaCCs and Transient Receptor Potential Vanilloid-4 (TRPV4) channels are co-expressed in some tissues where they functionally interact. TRPV4 is activated by different stimuli and forms a calcium permeable channel that is activated by GSK1016790A and antagonized by GSK2193874. Here we report that GSK2193874 enhances the chloride currents mediated by TMEM16B expressed in HEK cells at nanomolar concentrations and that GSK1016790A enhances native CaCCs of Xenopus oocytes. Thus, these compounds may be used as a tool for the study of CaCCs, TRPV4 and their interactions.

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Cloning and heterologous expression of new xANO2 from Xenopus laevis

Cited by 4 publications

References 20 publications

Isolation and Expression Profile of the Ca²⁺-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis

Isolation and Expression Profile of the Ca²⁺-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis

The Tmem16a chloride channel is required for mucin maturation after secretion from goblet-like cells in the Xenopus tropicalis tadpole skin.

Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

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Cloning and heterologous expression of new xANO2 from Xenopus laevis

Cited by 4 publications

References 20 publications

Isolation and Expression Profile of the Ca2+-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis

Isolation and Expression Profile of the Ca2+-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis

The Tmem16a chloride channel is required for mucin maturation after secretion from goblet-like cells in the Xenopus tropicalis tadpole skin.

Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

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Isolation and Expression Profile of the Ca²⁺-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis

Isolation and Expression Profile of the Ca²⁺-Activated Chloride Channel-like Membrane Protein 6 Gene inXenopus laevis