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, 284 (48), 33360-8

Regulation of TMEM16A Chloride Channel Properties by Alternative Splicing

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Regulation of TMEM16A Chloride Channel Properties by Alternative Splicing

Loretta Ferrera et al. J Biol Chem.

Abstract

Expression of TMEM16A protein is associated with the activity of Ca(2+)-activated Cl(-) channels. TMEM16A primary transcript undergoes alternative splicing. thus resulting in the generation of multiple isoforms. We have determined the pattern of splicing and assessed the functional properties of the corresponding TMEM16A variants. We found three alternative exons, 6b, 13, and 15, coding for segments of 22, 4, and 26 amino acids, respectively, which are differently spliced in human organs. By patch clamp experiments on transfected cells, we found that skipping of exon 6b changes the Ca(2+) sensitivity by nearly 4-fold, resulting in Cl(-) currents requiring lower Ca(2+) concentrations to be activated. At the membrane potential of 80 mV, the apparent half-effective concentration decreases from 350 to 90 nm when the segment corresponding to exon 6b is excluded. Skipping of exon 13 instead strongly reduces the characteristic time-dependent activation observed for Ca(2+)-activated Cl(-) channels at positive membrane potentials. This effect was also obtained by deleting only the second pair of amino acids corresponding to exon 13. Alternative splicing appears as an important mechanism to regulate the voltage and Ca(2+) dependence of the TMEM16A-dependent Cl(-) channels in a tissue-specific manner.

Figures

FIGURE 1.
FIGURE 1.
Alternative splicing pattern of TMEM16A. A, schematic representation of TMEM16A coding sequence structure showing the constitutive and alternative spliced exons in white and black boxes, respectively. Exons 6b, 13, and 15 are 66 bp, 12 bp, and 78 bp, respectively. The small black superimposed arrows represent the positions of the primers used for the RT-PCR analysis. B–D, splicing pattern of exons 6b, 13, and 15, respectively. The upper part of each panel corresponds to the RT-PCR-amplified bands run on an agarose gel and stained with ethidium bromide; the lower graphs represent the percentage of exon inclusion for each tissue expressed as mean ± S.D. of three independent experiments done in duplicate. A panel of total human RNAs (20 different normal human tissues) was analyzed with the following primers: 803D and 1385R for exon 6b, 1368D and 1525R for exon 13, and 1506D and 1894R for exon 15. The identity of the transcripts was verified by direct sequencing, and the inclusion (ex+) or exclusion forms (ex−) are indicated. M, molecular 1-kb marker.
FIGURE 2.
FIGURE 2.
Anion transport by TMEM16A isoforms. A, putative topology of the TMEM16A protein with the localization of the alternative segments a, b, c, and d. Inclusion/skipping of segments b, c, and d is due to alternative splicing of exons 6b, 13, and 15, respectively. Instead, skipping of segment a occurs by usage of an alternative promoter. B and C, representative traces (upper) and calculated I transport (lower) from experiments with the halide-sensitive YFP assay in transiently transfected HEK-293 cells. Cells expressing the indicated TMEM16A constructs were exposed to extracellular I (arrows) in the absence or presence of 1 μm ionomycin. The Ca2+ elevation triggered by ionomycin evoked a strong increase in I transport as evident from the sharp decrease in cell fluorescence. *, p < 0.05; **, p < 0.01 versus mock-transfected cells (n = 5–10).
FIGURE 3.
FIGURE 3.
Properties of TMEM16A-dependent currents in HEK-293 cells. A, whole cell membrane currents in HEK-293 cells transiently transfected with the indicated isoforms and mutants. Membrane currents were elicited by stepping membrane potential from −100 to 100 mV in 20-mV increments from a holding potential of −60 mV. Dotted lines indicate the zero-current level. The intracellular free Ca2+ concentration was 305 nm. B, current-voltage relationships determined for the various isoforms and mutants. Current amplitudes were measured at the end of test pulses. pF, picofarads. C, rectification index determined as the ratio of the current measured at +100 mV to the current measured at −100 mV. *, p < 0.05; **, p < 0.01 versus (abc) isoform. D, normalized instantaneous current (i.e. the ratio of the current at the beginning to that at the end of the voltage pulse). **, p < 0.01 versus (abc) isoform. Data in B–D are mean ± S.E. (n = 12–26).
FIGURE 4.
FIGURE 4.
Detection of TMEM16A protein by immunofluorescence. FRT cells with stable expression of TMEM16A isoforms (abc), (ac), and (ab) or null FRT cells were immunostained with an antibody against the TMEM16A protein (green). The nuclei were counterstained in blue. The images are representative of three separate experiments.
FIGURE 5.
FIGURE 5.
Membrane currents elicited by (abc) and (ac) isoforms. A and B, whole cell membrane currents recorded at the indicated intracellular Ca2+ concentrations in FRT cells with stable expression of (abc) and (ac) variants, respectively. Currents were evoked with the same voltage protocol described for Fig. 3. Dotted lines are the zero-current level. C and D, current-voltage relationships for the two isoforms at the indicated intracellular Ca2+ concentrations. pF, picofarads. Current amplitudes were measured at the end of test pulses (mean ± S.E., n = 10–31).
FIGURE 6.
FIGURE 6.
Ca2+ sensitivity of (abc) and (ac) isoforms. A, plot of membrane conductance (calculated from the tail currents at −60 mV) versus Ca2+ concentration for the two isoforms at various test potentials as indicated. nS/pF, nanosiemens/picofarads. Data obtained from experiments like those shown in Fig. 5 were fitted with the Hill equation to calculate the apparent dissociation constant, Kd. B, plot of Kd values for the two TMEM16A isoforms at various membrane potentials. C, plot of membrane conductance for (abc) and (ac) at two Ca2+ concentrations (115 and 305 nm) versus membrane potential. Data are mean ± S.E. (n = 10–31).
FIGURE 7.
FIGURE 7.
Properties of TMEM16(ab). A, representative whole cell membrane currents recorded in FRT cells with stable expression of TMEM16A(ab) at the indicated intracellular Ca2+ concentrations. B, current-voltage relationships from similar experiments (mean ± S.E., n = 8–13). pF, picofarads. C, normalized instantaneous current at 100 mV (determined as in Fig. 3D) for TMEM16(ab) compared with the (abc) isoform. Asterisks indicate a significant difference (p < 0.01). D, Ca2+ sensitivity of the (ab) isoform. Plot of membrane conductance versus Ca2+ concentration at the indicated membrane potentials is shown (mean ± S.E., n = 8–13).

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