Translocatable voltage-gated Ca 2+ channel β subunits in α1-β complexes reveal competitive replacement yet no spontaneous dissociation.

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.
Competing Interests: The authors declare no conflict of interest.