Allosteric Modulation of Ca2+-Activated Cl- Channles TMEM16A by PIP2 and CAMKII

Abstract

Transmembrane member 16A (TMEM16A), a member of 10 TMEM16 family, has been shown to encode Ca2+-activated Cl- channels that generate chloride conductance in an intracellular Ca2+- and voltage-dependent manner. Recently many studies showed that TMEM16A channel activity can be regulated by PIP2, a phospholipid placed in the inner leaflet of plasma membrane. However, it is not clearly established yet how the membrane PIP2 modulates TMEM16A gating activity in molecular level. Here we present that intracellular ATP changes PIP2 sensitivity of TMEM16A channels through CAMKII-dependent phosphorylation of the channel proteins. Through site-directed mutagenesis, we found that serine 673 (S673) is a crucial phosphorylation position affecting channel gating and PIP2 sensitivity of the channels. We further identified that PIP2 interacts with arginine 486 (R486) in the first intracellular loop as a putative binding site. Notably, our simulation data show that the phosphorylation region causes the change of structure of PIP2-binding site in the distance, thereby altering PIP2 sensitivity. Together, our present study supports that PIP2 in the plasma membrane regulates the activity of TMEM16A channel and structure simulation allow us to understand mechanistic insight into allosteric modulation of TMEM16A channel by phosphorylation and membrane PIP2.