Allosteric modulation of alternatively spliced Ca

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.