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标题：Allosteric modulation of alternatively spliced Ca2 -activated Cl− channels TMEM16A by PI(4,5)P2 and CaMKII
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作者：Woori Ko ; Seung-Ryoung Jung ; Kwon-Woo Kim 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：48
页码：30787-30798
DOI：10.1073/pnas.2014520117
出版社：The National Academy of Sciences of the United States of America
摘要：Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl − channel activated by intracellular Ca 2 and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P 2 degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P 2 depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P 2 sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca 2 /calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current ( i ), but not of channel number ( N ) or open probability ( P O ). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P 2 , and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P 2 . In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P 2 -binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P 2 , modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.