Cloning and heterologous expression of new xANO2 from Xenopus laevis

Rae Hyung Ryu, Soo Jin Oh, Ra Mi Lee, Seong Won Jeong, Lily Yeh Jan, Chi Ho Lee, C. Justin Lee, Sang Min Jeong

    Research output: Contribution to journalArticlepeer-review

    3 Citations (Scopus)

    Abstract

    We have successfully isolated a novel anoctamin (xANO2), Ca2+-activated chloride channel (ANO1, TMEM16A), from Xenopus laevis. The cDNA sequence was determined to belong to the anoctamin family by comparison with the xTMEM16A sequence in a previous report. Full length cDNA synthesis was performed by repeating 5'- and 3'-rapid amplification of cDNA end (RACE). We successfully completed the entire cDNA sequence and transiently named this sequence xANO2. The xANO2 cDNA is 3884 base pair (bp) long and codes 980 amino acid (aa) proteins. According to an aa homology search using the Basic Local Alignment Search Tool (BLAST), xANO2 showed an overall identity of 92% to xTMEM16A (xANO1) independently sub-cloned in our laboratory. A primary sequence of xANO2 revealed typical characteristics of transmembrane proteins. In tissue distribution analysis, the gene products of anoctamins were ubiquitously detected by real-time PCR (RT-PCR). The expression profiles of each anoctamin were different among brain, oocytes, and digestive organs with relatively weak expression. To clarify the anoctamin activity, physiological studies were performed using the whole cell patch-clamp technique with HEK293T cells, enhanced green fluorescent protein (EGFP), and expression vectors carrying anoctamins. Characteristics typical of voltage-dependent chloride currents were detected in cells expressing both xANO2 and xTMEM16A but not with EGFP alone. Sensitive reactions to the anion channel blocker niflumic acid (NFA) were also revealed. Considering these results, xANO2 was regarded as a new TMEM16A belonging to the Xenopus anoctamin family.

    Original languageEnglish
    Pages (from-to)559-565
    Number of pages7
    JournalBiochemical and biophysical research communications
    Volume408
    Issue number4
    DOIs
    Publication statusPublished - 2011 May 20

    Bibliographical note

    Funding Information:
    This research was supported by funding to S.M. Jeong from a Korean Research Foundation Grant by the Ministry of Education and Human Resources Development ( KRF-313-2007-2-E00519 ) and the Brain Korea 21 Project. This research was supported by the Technology Development Program for Agriculture and Forestry ( 2010-A008-0034 ) from the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea . The company grant was supported by Takara Korea. Xenopus laevis was kindly provided by Dr. Nah S.Y. (Konkuk University, Seoul, Korea).

    Keywords

    • Anoctamin gene
    • Calcium-activated chloride channel
    • Heterologous expression
    • Tissue distribution
    • Xenopus laevis

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology
    • Cell Biology

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