Abstract
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.
Highlights
IntroductionTMEM16A ( known as Anoctamin 1) is a Ca2+ -activated Cl− channel (CaCC) that is activated in response to intracellular Ca2+ increases and membrane depolarization [1,2,3]
This study expands our understanding of PI(4,5)P2 regulation of the TMEM16A channels in living cells
Using electrophysiological recordings and confocal microscopy, the present works show that two splice variants, TMEM16A TMEM16A(a) and TMEM16A(ac), are differentially regulated by the membrane lipid PI(4,5)P2
Summary
TMEM16A ( known as Anoctamin 1) is a Ca2+ -activated Cl− channel (CaCC) that is activated in response to intracellular Ca2+ increases and membrane depolarization [1,2,3]. The cytoplasmic N- and C-termini and the intracellular loops of TMEM16A possess critical regions that influence channel regulation, including dimerization [6], phosphorylation [7], and interactions with phosphoinositides such as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 ] [8,9,10]. There are four exon segments—a (116 residues), b (22 residues), c (4 residues), and d (26 residues)—in the N-terminus and first intracellular loop of TMEM16A. It is well established that several alternatively spliced TMEM16A isoforms are generated by the combinational exclusion or inclusion of each exon [11]. In addition to the four segments, the formation of other minor isoforms by Exons 1, 10, 14, and 18 has been reported [12]
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