+ Translocatable voltage-gated Ca2 channel β subunits in α1–β complexes reveal competitive replacement yet no spontaneous dissociation Jun-Hee Yeona, Cheon-Gyu Parka, Bertil Hilleb,1, and Byung-Chang Suha,1 aDepartment of Brain & Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, 42988 Daegu, South Korea; and bDepartment of Physiology and Biophysics, University of Washington, Seattle, WA 98195 Contributed by Bertil Hille, August 28, 2018 (sent for review June 8, 2018; reviewed by Diane Lipscombe and Daniel L. Minor Jr.) 2+ K β β subunits of high voltage-gated Ca (CaV) channels promote cell- affinity ( d) of 5 to 20 nM depending on the subunit isoform surface expression of pore-forming α1 subunits and regulate chan- and experimental conditions (15–23). In intact cells, the stability nel gating through binding to the α-interaction domain (AID) in of the channel CaV α1–β complex is not well understood, but that the first intracellular loop. We addressed the stability of CaV α1B–β interaction is said to have lower apparent affinity and to be more interactions by rapamycin-translocatable Ca β subunits that allow dynamic than in vitro. For instance, injection of purified β2a V Xenopus drug-induced sequestration and uncoupling of the β subunit from protein into oocytes expressing CaV2.3 precoupled with Ca 2.2 channel complexes in intact cells. Without Ca α1B/α2δ1, all β1b subunits changed the fast inactivating currents to slowly V V K modified β subunits, except membrane-tethered β2a and β2e, are in inactivating currents over several hours (24). The apparent d –β the cytosol and rapidly translocate upon rapamycin addition to an- for the CaV2.3 1b interaction is reported to rise to several chors on target organelles: plasma membrane, mitochondria, or endo- hundred nanomolar inside cells, allowing dynamic exchange of α α δ the β subunit on α1E subunits and competition in the binding of plasmic reticulum. In cells coexpressing CaV 1B/ 2 1subunits,the β α translocatable β subunits colocalize at the plasma membrane with two different isoforms for one 1 subunit (25). The apparent α α1–β binding affinity depends on the molecular properties of β 1B and stay there after rapamycin application, indicating that inter- α –β α – actions between α1B and bound β subunits are very stable. However, isoforms; thus, the 1C 1a complex is more stable than 1C β α –β NEUROSCIENCE the interaction becomes dynamic when other competing β isoforms 2a or 1C 4 in intact skeletal muscle cells (26). Similarly, in- tracellular perfusion of degradation-protected AID peptides are coexpressed. Addition of rapamycin, then, switches channel gating blocked the interaction of CaV1.2 channels with β3 subunits, but and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] not with β2a, indicating that the CaV1.2–β3 complex is less stable lipid. Thus, expression of free β isoforms around the channel reveals –β α –β than the CaV1.2 2a complex inside cells (27). In contrast, in a dynamic aspect to the 1B interaction. On the other hand, excised inside-out patches, the β2a subunit remains stably bound translocatable β subunits with AID-binding site mutations are eas- to the core domains of CaV2.1 channels unless the binding af- ily dissociated from CaV α1B on the addition of rapamycin, decreas- finity of CaV β GK domain is reduced by mutating the α-binding ing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the pocket (ABP) site (28). Thus, in vitro work has emphasized high mutations slow CaV2.2 current inactivation and shift the voltage stability of CaV–β complexes whereas intact-cell work has em- dependence of activation to more positive potentials. Mutated phasized dynamic exchange of β subunits. β translocatable subunits work similarly in CaV2.3 channels. In sum, Here, we developed chimeric translocatable Ca β subunits. They α –β V the strong interaction of CaV 1B subunits can be overcome by can be translocated rapidly by chemically inducible heterodimerization other free β isoforms, permitting dynamic changes in channel prop- erties in intact cells. Significance 2+ β voltage-gated Ca channel | CaV subunits | chemically inducible 2+ Voltage-gated Ca (CaV) channels have an α1-α2δ core com- dimerization | rapamycin | PI(4,5)P2 plexed with one of several alternative β subunits. Contradictory i β 2+ evidence says that, once bound, ( )a subunit is permanently oltage-gated Ca (CaV) channels play essential roles con- α α δ ii 2+ associated with the 1- 2 core or ( )thatitisfreetobe Vverting electrical signals to changes in Ca -dependent pro- exchanged for other β subunits. We designed rapamycin- cesses like synaptic transmission, muscle contraction, and gene translocatable CaV β subunits that allow drug-induced seques- transcription (1). The CaV channels can be divided into high voltage- tration of free β subunits to several organelle anchors. Seques- activated (HVA) (CaV1andCaV2) and low voltage-activated (LVA) tering free subunits does not dissociate bound subunits from (CaV3) channels in accordance with their activation threshold. HVA channels except when the binding site is mutated to weaken the 2+ α Ca channels are composed of a pore-forming 1 subunit and at interaction. Nevertheless, our rapamycin constructs show that, α least three auxiliary subunits, the disulfide-linked complex of 2and when nontranslocatable β subunits are coexpressed with a δ plus β. The auxiliary β subunit regulates cell surface trafficking and translocatable subunit, sequestering the translocatable subunit 2+ biophysical gating properties of HVA Ca channels via an in- changes the channel properties, revealing a quick replacement α – teraction with the CaV 1 subunit in 1:1 stoichiometry (2 4). Four by the nontranslocatable subunit in the channel complex. 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 Author contributions: J.-H.Y., C.-G.P., B.H., and B.-C.S. designed research; J.-H.Y. and HOOK domain separating highly conserved src homology-3 (SH3) C.-G.P. performed research; J.-H.Y., C.-G.P., and B.-C.S. analyzed data; and J.-H.Y., B.H., and guanylate kinase (GK) domains. The GK domain contains the and B.-C.S. wrote the paper. α-binding pocket (ABP) that interacts directly with the α-interaction Reviewers: D.L., Brown University; and D.L.M., University of California, San Francisco. The authors declare no conflict of interest. domain (AID) of the cytosolic I-II loop of CaV α1 subunits (8–11). An additional lower affinity binding site in the C terminus of β Published under the PNAS license. subunits contributes to the interaction with the C terminus of α1and 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. to regulation of channel-gating properties (12–14). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Studies with in vitro assays report that the interaction between 1073/pnas.1809762115/-/DCSupplemental. the AID of CaV α1 and CaV β subunits is strong, with a binding www.pnas.org/cgi/doi/10.1073/pnas.1809762115 PNAS Latest Articles | 1of10 Downloaded by guest on September 26, 2021 to a membrane anchor on an intracellular organelle, including CaV β GK domain (28). We show that acute dissociation of β the plasma membrane, mitochondria, and endoplasmic reticu- subunits from CaV α1B dramatically changes the functional lum (ER), upon addition of rapamycin. Using this method, rapid properties of CaV2.2 channels. sequestration of free β subunits helped us to analyze the stability of CaV α1–β complexes in intact cells and the roles of β subunits Results in channel gating. The heterodimerization is based on formation Generation and Characterization of Translocatable CaV β Subunits. of a ternary complex between recombinant FK506 binding pro- CaV β subunits are noncovalently coupled to membrane-resident tein (FKBP) and FKBP-rapamycin binding (FRB) protein upon CaV α1 subunits of living cells (32). To further understand the addition of rapamycin (29–31). In our experiments, chimeric β interaction properties, we used rapamycin-induced translocation subunit-FKBP proteins were heterodimerized through rapamy- of β subunits. CaV β isoforms were labeled at the C terminus with cin with an FRB anchor on a target organelle. The designed recombinant FKBP and then in tandem with green (or mCherry) translocatable CaV β systems permit study of association or dis- fluorescent protein; this produced the final β-FKBP-GFP (β-FG) sociation of β subunits from CaV α1B channels located in the proteins that can be recruited to specific subcellular target or- plasma membrane in living cells. We provide evidence that the ganelles by rapamycin (Fig. 1A and SI Appendix,Fig.S1). The interaction of CaV α1 with β subunits can be very stable inside target organelles were labeled with FRB and mCherry-fluorescent cells. The complex does not dissociate up to several hours. protein fused to organelle membrane-specific marker proteins: However, if there are other types of free β isoforms around the Lyn11 for the plasma membrane (LDR), Tom20 for mitochondrial channels, there can be exchange with the coupled β subunits on outer membranes (Tom20-MR), and Cb5 for the ER (MR-Cb5). the α1B protein. The CaV α1–β interaction can be weakened by FKBP and FRB construct pairs allowed rapid sequestration of mutating two residues in the α-binding pocket (ABP) site of the recombinant β-FG protein to specific organelles via the formation Fig. 1. Subcellular translocation of CaV β subunits on the addition of rapamycin in intact tsA-201 cells. (A) Schematic diagram of translocatable β subunit (β-FG) and target organelle anchors.