Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl⁻ channel activated by intracellular Ca²⁺ and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P₂ degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P₂ 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)P₂ sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca²⁺/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)P₂, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P₂. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P₂-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)P₂, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

跨膜 16A（TMEM16A，anoctamin1）是 10 种 TMEM16 家族蛋白中的一种，是一种由细胞内 Ca ²⁺和膜电压激活的 Cl ^-通道。该通道还受膜磷脂磷脂酰肌醇 4,5-二磷酸 [PI(4,5)P ₂ ] 的调节。我们发现TMEM16A的两个剪接变体对内源性PI(4,5)P ₂降解表现出不同的敏感性，其中TMEM16A(ac)比TMEM16A(a)表现出更高的通道活性和更多的PI(4,5)P ₂消耗电流抑制）。这两种通道亚型的不同之处在于 c 段（外显子 13）的选择性剪接。 TMEM16A(ac) 和 (a) 的电流振幅和 PI(4,5)P ₂敏感性因游离胞质 ATP 的减少和 Ca ²⁺ /钙调蛋白依赖性蛋白激酶 II (CaMKII) 磷酸化的减少而显着增强。 ）。噪声分析表明电流的增加是由于单通道电流 ( i ) 的增加，而不是通道数 ( N ) 或开路概率 ( P _O ) 的增加。诱变表明第一个细胞内环中的精氨酸 486 作为 PI(4,5)P ₂的推定结合位点，第三个细胞内环中的丝氨酸 673 作为调节 PI(4,5)P 作用的调节通道磷酸化位点。 5）P ₂ 。计算机模拟表明，S673 的磷酸化如何变构且不同地改变具有和不具有 c 段外显子的通道剪接变体之间的远处 PI(4,5)P ₂结合位点的结构。 总之，我们的研究揭示了以下内容：质膜 PI(4,5)P ₂对选择性剪接的 TMEM16A(ac) 和 (a) 的差异调节、通过通道磷酸化对这些效应的修改、分子位点的识别以及机制通过计算机模拟进行解释。