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Biochem Biophys Res Commun
2014 Apr 18;4464:1096-101. doi: 10.1016/j.bbrc.2014.03.057.
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TMEM16A alternative splicing isoforms in Xenopus tropicalis: distribution and functional properties.
Huanosta-Gutiérrez A
,
Espino-Saldaña AE
,
Reyes JP
,
Pétriz A
,
Miledi R
,
Martínez-Torres A
.
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Oocytes of Xenopus tropicalis elicit a Ca(2+)-dependent outwardly rectifying, low-activating current (ICl,Ca) that is inhibited by Cl(-) channel blockers. When inactivated, ICl,Ca shows an exponentially decaying tail current that is related to currents generated by TMEM16A ion channels. Accordingly, RT-PCR revealed the expression of five alternatively spliced isoforms of TMEM16A in oocytes, which, after expression in HEK-293 cells, gave rise to fully functional Cl(-) channels. Upon hyperpolarization to -80 mV a transient current was observed only in isoforms that carry the exon 1d, coding for two potentially phosphorylatable Threonine residues. The identified isoforms are differentially expressed in several tissues of the frog. Thus, it appears that X. tropicalis oocytes express TMEM16A that gives rise to a Ca(2+)-dependent Cl(-) current, which is different from the previously reported voltage-dependent outwardly rectifying Cl(-) current.
Fig. 1.
Properties of CaCC natively expressed in X. tropicalis oocytes. (A–D) Representative current traces upon application of the voltage protocol described in Section 2 from oocytes bathed in external solution containing 10 mM Ca2+ or the indicated blockers (300 μM). (E) Dose–response curve for each channel blocker (n = 5; N = 4). Current is normalized. Data points are means ± SEM.
Fig. 2.
Alternatively spliced variants of xtTMEM16A. (A) Topological model of xtTMEM16A. The four segments that can be alternatively spliced are shown in red. The amino acid sequence of exon 1d is shown as an inset. The Thr residues indicated in red are potential targets for phosphorylation. (B) Distribution of xtTMEM16A. Nine different tissues and oocytes were screened for the expression of xtTMEM16A exons 1b, 1d, 13, and 15. Sk Mus, Skeletal muscle; Sm Int, small intestine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3.
(A) GFP-fused xtTMEM16A (13) partially co-localized with FM4-64 in the plasma membrane of transfected HEK cells (arrows); GFP fluorescence did not localize on the cell surface (arrowheads). Orthogonal view (yellow lines) outline the differences in the GFP and xtTMEM16A (13). Scale bar = 5 μm (B) Sample currents of the five isoforms showing the time courses of current activation and deactivation upon application of the voltage protocol described in Section 2. The red arrows indicate the amplitudes of the current measured at the start (Istart), −100 mV, and the current measured at the end (Iend), −100 mV, used to calculate the ratio (Istart/Iend). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
