Molecular mechanisms for allosteric modulation of alternatively spliced Ca2+-activated Cl- channels TMEM16A by PI(4,5)P2 and CaMKII

Abstract

Transmembrane 16A (TMEM16A, anoctamin1), one of ten 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). I found 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). I further investigated biophysical properties for PI(4,5)P2 regulation using TMEM16A(ac). The PI(4,5)P2 sensitivity of TMEM16A(ac) shows outstanding effects in the slow current of TMEM16A activation steps. The experiments through rapamycin-inducible dimerization provides reasonable evidence that is voltage-independent PI(4,5)P2 regulation on TMEM16A channel. These inhibitory effects of PI(4,5)P2 depletion abolished when TMEM16A is co-expressed with PIP 5-kinase type-1γ (PIPKIγ) increasing PI(4,5)P2 levels. In addition, I confirmed PI(4,5)P2 resynthesis is essential for TMEM16A recovery from PI(4,5)P2 depletion-induced current inhibition. These data suggest that TMEM16A(ac) needs membrane lipid PI(4,5)P2, as a cofactor, for channel gating. Furthermore, this study implies that studying PI(4,5)P2 regulation on other TMEM16’s splice variants is worth helping to understand the function of each segment physiologically.