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Biochem Biophys Res Commun
2011 May 20;4084:559-65. doi: 10.1016/j.bbrc.2011.04.060.
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Cloning and heterologous expression of new xANO2 from Xenopus laevis.
Ryu RH
,
Oh SJ
,
Lee RM
,
Jeong SW
,
Jan LY
,
Lee CH
,
Lee CJ
,
Jeong SM
.
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We have successfully isolated a novel anoctamin (xANO2), Ca(2+)-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.
Fig. 1.
Construction strategy of full-length cDNA. (A) To obtain the full length of xANO2 cDNA, three rounds of PCR were carried out under appropriate conditions. Initial cDNA (dotted line) and PCR products (thick line) are depicted with black lines. 3â²-RACE products are represented as Round 1 and 5â²-RACE products are represented as Round 2 and 3. (B) PCR products of 3â²-RACE (left, Round 1) and 5â²-RACE (middle: Round 2, right: Round 3) were electrophoresed in a 1% agarose gel. Black arrowheads indicate the 2.0, 0.7, and 1.7 kb target products. (C) Full-length PCR product of X. laevis TMEM16A gene. The 3012 base pair (bp) cDNA (xTMEM16A) was amplified by gene specific primers: CAN3 and CAN5.
Fig. 2.
Primary structure analysis of X. laevis anoctamins. (A) Amino acid (aa) alignment was deduced by querying the xANO2 aa sequence using GENETYX. The aa homology is indicated as follows: identical (asterisk), similar (dot). The putative transmembrane regions are marked by an upper line. Consensus sites are marked for N-linked glycosylation (â), phosphorylation by PKA (â¾), PKC (+), PKG (â ), casein II (#). (B) Hydrophobicity plots (KyteâDoolittle analysis) of the anoctamin aa sequences. Both hydropathy plots were absolutely consistent in the overall region except for a short range at the amino terminus. The hydropathy plots were analyzed using an analysis window of 17 residues, and then overlapped. Hydrophobic domains are given as positive values. The proposed TM segments and reentrant loop are indicated by horizontal lines and dotted line, respectively.
Fig. 3. Comparison of anoctamin expression in X. laevis by reverse transcriptase (RT)-PCR and real-time PCR. Each expression level of anoctamins was normalized with β-actin used as internal control. (A) Bar graphs of RT-PCR. One round of PCR was carried out with 38 repeats to determine the specific products of xANO2, xTMEM16A, and β-actin gene. Each specific product of xANO2, xTMEM16A, and β-actin gene was amplified by RT-PCR. The β-actin gene was used as an internal control. Lanes 1–9 represent brain, heart, lung, liver, small intestine, colon, kidney, spleen and oocyte, respectively. (B) Quantitative histogram of real-time PCR. Real-time PCR was carried out with 50 repeats for quantitative analysis for anoctamin expression in X. laevis tissues. (C) Tissue distributions of anoctamin mRNA expression. Quantitation of anoctamin is shown as a bar graph compared to RT-PCR.
Fig. 4.
xANO2 as a Ca2+ activated anion channel. (A and C) Representative IâV responses of HEK293T cells expressing xTMEM16A (A), xANO2 (C), or mock control under whole-cell patch-clamp configuration using 4.5 μM Ca2+-containing patch pipette solution. (B and D) Bar graph showing summary of current amplitudes recorded from a holding potential of â100 to +100 mV (mean ± SEM). The anion channel blocker niflumic acid (NFA; 100 μM) was preincubated with cells for 10 min. Number of determinations are indicated on the bar graph. âââp < 0.0001 vs. anoctamin-expressing cells with 4.5 μM Ca2+ pipette solution, two tailed t-test. Each B and D bar graphs is depicted for A and C IâV responses. (E) Relative ratios of current amplitude. Each bar was depicted by the calculated percentage for xTMEM16A as full positive current.
