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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)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). The current amplitude and PI(4,5)P2 sensitivity 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 (P O). 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.

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl(−) channel activated by intracellular Ca(2+) and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P(2) degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P(2) depletion 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)P(2) sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca(2+)/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 (P(O)). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P(2), and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P(2). In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P(2)-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)P(2), modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.

β subunits of high voltage-gated Ca(2+) (Ca(V)) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of Ca(V) α1B–β interactions by rapamycin-translocatable Ca(V) β subunits that allow drug-induced sequestration and uncoupling of the β subunit from Ca(V)2.2 channel complexes in intact cells. Without Ca(V) α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing Ca(V) α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B–β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from Ca(V) α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P(2) sensitivity. Furthermore, the mutations slow Ca(V)2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in Ca(V)2.3 channels. In sum, the strong interaction of Ca(V) α1B–β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.

The PI(4,5)P2 level in the plasma membrane is dynamically regulated by cytoplasmic ATP production and receptor-mediated transmembrane signaling cascades. In this issue of Cell Chemical Biology, Xie et al. (2016) use optogenetics to micro-manipulate membrane PI(4,5)P2 and reveal how acute PI(4,5)P2 changes can alter intracellular Ca(2+) dynamics and insulin secretion in pancreatic β cells.

High-voltage-gated Ca2+ (CaV) channels consist of a pore-forming α1 subunit and two auxiliary α2δ and β subunits. Although it is well established that CaV β promotes cell surface expression and regulates the gating properties of CaV channels, the stability of the α1-β interaction in vivo remains unclear. Here, we address this issue by engineering translocatable CaV β systems that allow the real-time measurement of the coupling in live imaging and patch-clamp. In cells without CaV α1B expression, all constructed β subunits except palmitoylated β2a were translocated to the intracellular target membranes by rapamycin application. However, in cells co-expressed with CaV α1B no translocation of the β subunits was measured up to 2 hrs. In addition, rapamycin-induced recruitment of CaV β subunits to the plasma membrane did not affected the gating properties of CaV channels. In contrast, double mutation of CaV β subunits was shown to be dissociated easily from CaV α1 by rapamycin application. In these mutant forms, dissociation of β subunit from CaV α1B lead to the decrease in current amplitude, PIP2 sensitivity and current recovery from Gβγ inhibition. Furthermore, it inhibited inactivation and shifted the voltage dependent IV curve to the right in the live cells. When cells were cotransfected with double mutated CaV β1b and β2a together, rapamycin lead to dissociation of β1b, but not β2a, from CaV α1B. The CaV α1B separated from β1b did not further interact with CaV β2a, suggesting that once dissociated, CaV α1 do not interact with other β. Taken together, our data demonstrate that the interaction of CaV α1 with β subunit is very stable in live cells.

Voltage-gated calcium (CaV) channel β subunit increases the expression of pore-forming α1 subunit in the plasma membrane and regulates the biophysical properties of channel gating. In particular, the gating of high-voltage activated (HVA) Ca2+ channels is differentially controlled by CaV β subunits depending on the isotypes and intracellular location. However, the molecular mechanism of type specificity and cellular location of β subunit in CaV channel regulation is not clearly determined. We confirmed the constitutive localization of β2a in the plasma membrane irrelevant of the existence of α1B and α2δ1 is due to palmitoylation of N-terminus, and that CaV2.2 current with β2a is slowly inactivated compare to the cells with other types of cytosolic β subunits. In order to further understand the functional role of palmitoylation in the formation of channel complex and current regulation, we constructs a translocatable β2a(C3,4S)-FG a palmitoylation-resistant form of β2a, which is by tagged the β2a(C3,4S) with a FKBP domain and a green fluorescent protein (GFP) sequentially on its C-terminus as a chemically-inducible dimerization (CID) system. When the mutant β2a(C3,4S)-FG was expressed in tsA201 cells, it was mainly located in the plasma membrane in the presence of α1B, while it distributes throughout the cytosol without the α1B. Since the palmitoylation does not occur in the mutant form, the targeting of β2a(C3,4S)-FG to the plasma membrane can be only due to the interaction between α1B and β subunits. We also found that β2a(C3,4S)-FG and β2b-FG are not translocated to the cytosolic organelles by the application of rapamycin. Thus, the results demonstrate that the interaction between α1B and β2a subunits is irreversible.