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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.