Allosteric modulation of alternatively spliced Ca²⁺-activated Cl⁻channels TMEM16A by PI(4,5)P₂and CaMKII

Abstract

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 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]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P2degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2depletion 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)P2sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca2+/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 (PO). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P2, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P2. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P2-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)P2, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

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

“…Membrane PI(4,5)P 2 would be more important for maintaining the channels in the fully open state with two Ca 2+ -binding sequences. Similarly, we report that the modulatory effects of PI(4,5)P 2 are stronger in unphosphorylated TMEM16A(ac) channels [ 10 ], suggesting that the binding of PI(4,5)P 2 to TMEM16A channels is dynamically regulated by allosteric changes in the channel structure.…”

“…Our data showed how membrane PI(4,5)P 2 turnover modulates the TMEM16A channels in living cell membranes and revealed novel differences between the two isoforms of TMEM16A channels in the modulation by PI(4,5)P 2 . Our recent study revealed that the c-segment allosterically regulates the interaction of TMEM16A channels with PI(4,5)P 2 by altering the structure of PI(4,5)P 2 -binding sites on the channel protein, while it does not bind to PI(4,5)P 2 directly [ 10 ]. The activity of the TMEM16A(ac) channels is selectively decreased by the conversion of PI(4,5)P 2 to PI4P and remains inhibited until PI(4,5)P 2 is resynthesized.…”

“…The TMEM16A channel is a homodimeric protein complex consisting of two identical subunits with 10 transmembrane domains (TMDs). 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)P 2 ] [ 8 , 9 , 10 ]. In particular, 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.…”

“…In fact, the first intracellular TM2–TM3 loop contains several important regulatory domains such as the c-segment and PI(4,5)P 2 -binding region. In our recent study, we found that those PI(4,5)P 2 binding sites in TMEM16A are allosterically modulated by the distant phosphorylation of S673 by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) [ 10 ].…”

Ca2+ Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca2+-Binding Reservoir at the N-Terminal Domain

“…ANO6-1-6 showed increased Ca 2+ sensitivity compared with that of ANO6, but it was not as high as that of ANO1, which is around 1 μM ( Pedemonte and Galietta, 2014 ; Tak et al, 2019 ; Yang et al, 2008 ). Other unknown mechanisms may be accountable for the remaining difference in Ca 2+ sensitivity, such as additional regulatory sites existing only in ANO1, different interactions with phospholipids, or unknown allosteric effects ( Feng et al, 2019 ; Ko et al, 2020 ; Xiao et al, 2011 ; Ye et al, 2018 ; Yu et al, 2019 ). Further studies are needed to fully understand the exact mechanisms.…”