Regulation of TMEM16A Chloride Channel Properties by Alternative Splicing
Regulation of TMEM16A Chloride Channel Properties by Alternative Splicing
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DOI：
10.1074/jbc.m109.046607
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发表时间：
2009-11-27
影响因子：
4.8
通讯作者：
Galietta, Luis J. V.
中科院分区：
文献类型：
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作者：
Ferrera, Loretta;Caputo, Antonella;Galietta, Luis J. V.
TMEM 16 A蛋白的表达与Ca ~（2+）激活的Cl ~-通道的活性有关。TMEM 16 A初级转录物经历选择性剪接。从而导致产生多种同种型。我们已经确定了剪接的模式，并评估了相应的TMEM 16 A变体的功能特性。我们发现了三个替代外显子，6 b，13和15，分别编码22，4和26个氨基酸的片段，它们在人体器官中被不同地剪接。通过对转染细胞的膜片钳实验，我们发现外显子6 b的跳跃改变了近4倍的Ca 2+敏感性，导致Cl-电流需要较低的Ca 2+浓度才能被激活。在80 mV的膜电位下，当排除对应于外显子6 b的片段时，表观半有效浓度从350 nM降低至90 nM。外显子13的跳跃反而强烈降低了在正膜电位下观察到的Ca 2+激活的Cl-通道的特征性时间依赖性激活。通过仅删除对应于外显子13的第二对氨基酸也获得了这种效果。选择性剪接似乎是以组织特异性方式调节TMEM 16 A依赖性Cl-通道的电压和Ca 2+依赖性的重要机制。
Expression of TMEM16A protein is associated with the activity of Ca2+-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 Ca2+ sensitivity by nearly 4-fold, resulting in Cl- currents requiring lower Ca2+ 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 Ca2+-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 Ca2+ dependence of the TMEM16A-dependent Cl- channels in a tissue-specific manner.