Membrane targeting mechanism of voltage-gated calcium channel β subunits

Abstract

High voltage-gated Ca2+ (CaV) channels are protein complexes containing α1, β, and α2δ subunits. Although α1 subunit determines primarily gating property of CaV channels and their pharmacological response, as one of auxiliary subunits, β subunit is key regulator for CaV channel gating and receptor modulation. In particular, their subcellular localization is important for regulation of current inactivation and lipid-sensitivity of CaV channels. Most of the β subunit isoforms are cytosolic proteins, whereas two isoforms β2a and β2e subunits are localized in the plasma membrane. β2a is expressed in the plasma membrane via palmitoylation on two cystein residues of N-terminal region whereas membrane targeting mechanism of β2e remains unclear. Here we investigated how β2e is associated with the plasma membrane and what mechanism triggers reversible translcoation of β2e. Furthermore, we explored how such features have an influence on properties of CaV channels in terms of pore gating and lipid modulation. Through mutagenesis and liposome binding assays, we determined that the N-terminus, which contains seven basic residues and a hydrophobic residue, is responsible for this membrane tethering via electrostatic interaction with the plasma membrane. Particularly, we found that Lys2 (K2) and Trp5 (W5) proximal to N-terminus play a pivotal role in membrane association of β2e. Using rapamycin-inducible dimerization system, β2e exhibited cytosolic distribution when membrane phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PIP2) were simultaneously depleted by membrane recruitment of PJ, which is consisting of 4-phosphatase (sac) and 5-phosphatase (INPP5E). Additionally, in M1 muscarinic receptor (M1R) expressing cells, muscarinic stimulation induced translocation of β2e. In consistent with dynamic location change of β2e, in current recording, CaV channels displayed accelerated inactivation when β2e was localized in the cytosol. In experiments with Danio rerio voltage-sensitive phosphatase (Dr-VSP) for PIP2 depletion, out data show that current inhibition with K2A or W5A was 19% by PIP2 depletion. In addition, a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP2 sensitivity of CaV2.2 and CaV1.3 channels by ~2-fold. The results suggest that membrane targeting of the β2e subunit is mediated by nonspecific electrostatic insertion and dynamically regulated by receptor stimulation. Together, the phospholipid-protein interaction observed here provides structural insight into general principles for membrane-protein association and regulatory roles of phospholipids in ion channels. Next, we investigated the involvement of second messengers, such as protein kinases and calmodulin (CaM), in translocation of β2e to the cytosol since GPCR activation triggers various signaling molecules such as calmodulin and protein kinases. Using diverse approaches, our findings indicates that rise in cytosolic Ca2+ induces translocation of the β2e, and such shuttling is directly regulated by Ca2+ rather than other second messengers such protein kinases and CaM due to Ca2+ mediated screening effect. In addition, membrane dissociation of the β2e by rise in cytosolic Ca2+ accelerated inactivation of CaV channel even in the presence of Ca2+-insensitive CaM and augmented PIP2 sensitivity to CaV channel. Thus, we suggest that Ca2+ is a determinant for dynamic translocation of the β2e and profoundly affects gating property of CaV channels via dynamics regulation of the β2e. These results reveal a role of Ca2+ on ionic protein-lipid interaction and provide a novel regulatory mechanism of CaV channels. Collectively, we show for the first time that Ca2+ ions directly regulate the CaV channel gating through the control of membrane tethering of β2e subunit and the dynamic interplay between β2e subunit and cytosolic Ca2+ constitutes a novel feedback mechanism for the regulation of CaV channels. ⓒ 2016 DGIST