Differential Regulation of Ca2+-Activated Cl− Channel TMEM16A Splice Variants by Membrane PI(4,5)P2

TMEM16A is a Ca²⁺-activated Cl⁻ channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP₂ in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl⁻ currents, which are independent of the membrane potential and specific to PI(4,5)P₂ depletion. The attenuation of endogenous PI(4,5)P₂ levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl⁻ currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P₂, not PI4P and PI(3,4,5)P₃. Finally, we also confirmed that PI(4,5)P₂ resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P₂ selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.