β subunits of high voltage-gated Ca 2+ (Ca V ) channels promote cellsurface 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.voltage-gated Ca 2+ channel | Ca V β subunits | chemically inducible dimerization | rapamycin | PI(4,5)P 2 V oltage-gated Ca 2+ (Ca V ) channels play essential roles converting electrical signals to changes in Ca 2+ -dependent processes like synaptic transmission, muscle contraction, and gene transcription (1). The Ca V channels can be divided into high voltageactivated (HVA) (Ca V 1 and Ca V 2) and low voltage-activated (LVA) (Ca V 3) channels in accordance with their activation threshold. HVA Ca 2+ channels are composed of a pore-forming α1 subunit and at least three auxiliary subunits, the disulfide-linked complex of α2 and δ plus β. The auxiliary β subunit regulates cell surface trafficking and biophysical gating properties of HVA Ca 2+ channels via an interaction with the Ca V α1 subunit in 1:1 stoichiometry (2-4). Four distinct genes encode β1-β4 subunits and their splice variants (5-7). The β subunits contain a highly variable N and C terminus and a HOOK domain separating highly conserved src homology-3 (SH3) and guanylate kinase (GK) domains. The GK domain contains the α-binding pocket (ABP) that interacts directly with the α-interaction domain (AID) of the cytosolic I-II loop of Ca V α1 subunits (8-11). An addition...