Fig. 2. Alternative splicing of exon 14 of ano2 alters activation kinetics and calcium sensitivity. (A) The RNA/cDNA sequence of isoform 1 (in black) and 2 (in blue) of ANO2 channels are depicted, and exon 14 is marked in an orange box. (B) RT-PCR analysis of the stable HEK293T cell lines with or without the exon 14 (isoform 1 and 2, respectively) and the untransfected HEK293T cell for the control RT-PCR. Primers were designed to report the variants containing the common exon (exon 24) and exon 14. (C) Quantitative RT-PCR analysis for ano2 with common exon and exon 14 from the stable cell lines expressing isoform 1 and 2. (D) Representative traces of non-stationary currents of isoforms 1 and 2 measured at 0, 1, and 5 μM [Ca2+]i from stable HEK293T cell lines. Ca2+ was applied in the intrapipette solution. The holding potential was -70 mV. (E) The non-stationary currents were analyzed for the time constant (τactivation) of activation. (F) The τactivation of isoform 1 and isoform 2. (G) Representative traces of non-stationary currents of isoforms 1 and 2 recorded at 10~500 μM [Ca2+]i. (H) Regression between intracellular calcium concentration and τactivation in each isoform. (I) The dose–response relationship was calculated from the currents at 1~500 intracellular calcium. Lines were fitted to the Hill equation. Error bars indicate SEM. *p<.05 and ***p<.001; ns, not significant.
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