Translocatable voltage-gated Ca2+ channel β subunitsin α1–β complexes reveal competitive replacement yetno spontaneous dissociationJun-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 ofPhysiology 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.)

β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate chan-nel gating through binding to the α-interaction domain (AID) inthe first intracellular loop. We addressed the stability of CaV α1B–βinteractions by rapamycin-translocatable CaV β subunits that allowdrug-induced sequestration and uncoupling of the β subunit fromCaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, allmodified β subunits, except membrane-tethered β2a and β2e, are inthe cytosol and rapidly translocate upon rapamycin addition to an-chors on target organelles: plasma membrane, mitochondria, or endo-plasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, thetranslocatable β subunits colocalize at the plasma membrane withα1B and stay there after rapamycin application, indicating that inter-actions between α1B and bound β subunits are very stable. However,the interaction becomes dynamic when other competing β isoformsare coexpressed. Addition of rapamycin, then, switches channel gatingand regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]lipid. Thus, expression of free β isoforms around the channel revealsa dynamic aspect to the α1B–β interaction. On the other hand,translocatable β subunits with AID-binding site mutations are eas-ily dissociated from CaV α1B on the addition of rapamycin, decreas-ing current amplitude and PI(4,5)P2 sensitivity. Furthermore, themutations slow CaV2.2 current inactivation and shift the voltagedependence of activation to more positive potentials. Mutatedtranslocatable β subunits work similarly in CaV2.3 channels. In sum,the strong interaction of CaV α1B–β subunits can be overcome byother free β isoforms, permitting dynamic changes in channel prop-erties in intact cells.

voltage-gated Ca2+ channel | CaVβ subunits | chemically inducibledimerization | rapamycin | PI(4,5)P2

Voltage-gated Ca2+ (CaV) channels play essential roles con-verting electrical signals to changes in Ca2+-dependent pro-

cesses like synaptic transmission, muscle contraction, and genetranscription (1). The CaV channels can be divided into high voltage-activated (HVA) (CaV1 and CaV2) and low voltage-activated (LVA)(CaV3) channels in accordance with their activation threshold. HVACa2+ channels are composed of a pore-forming α1 subunit and atleast three auxiliary subunits, the disulfide-linked complex of α2 andδ plus β. The auxiliary β subunit regulates cell surface trafficking andbiophysical gating properties of HVA Ca2+ channels via an in-teraction with the CaV α1 subunit in 1:1 stoichiometry (2–4). Fourdistinct genes encode β1-β4 subunits and their splice variants (5–7).The β subunits contain a highly variable N and C terminus and aHOOK domain separating highly conserved src homology-3 (SH3)and guanylate kinase (GK) domains. The GK domain contains theα-binding pocket (ABP) that interacts directly with the α-interactiondomain (AID) of the cytosolic I-II loop of CaV α1 subunits (8–11).An additional lower affinity binding site in the C terminus of βsubunits contributes to the interaction with the C terminus of α1 andto regulation of channel-gating properties (12–14).Studies with in vitro assays report that the interaction between

the AID of CaV α1 and CaV β subunits is strong, with a binding

affinity (Kd) of 5 to 20 nM depending on the β subunit isoformand experimental conditions (15–23). In intact cells, the stabilityof the channel CaV α1–β complex is not well understood, but thatinteraction is said to have lower apparent affinity and to be moredynamic than in vitro. For instance, injection of purified β2aprotein into Xenopus oocytes expressing CaV2.3 precoupled withβ1b subunits changed the fast inactivating currents to slowlyinactivating currents over several hours (24). The apparent Kdfor the CaV2.3–β1b interaction is reported to rise to severalhundred nanomolar inside cells, allowing dynamic exchange ofthe β subunit on α1E subunits and competition in the binding oftwo different β isoforms for one α1 subunit (25). The apparentα1–β binding affinity depends on the molecular properties of βisoforms; thus, the α1C–β1a complex is more stable than α1C–β2a or α1C–β4 in intact skeletal muscle cells (26). Similarly, in-tracellular perfusion of degradation-protected AID peptidesblocked the interaction of CaV1.2 channels with β3 subunits, butnot with β2a, indicating that the CaV1.2–β3 complex is less stablethan the CaV1.2–β2a complex inside cells (27). In contrast, inexcised inside-out patches, the β2a subunit remains stably boundto the core domains of CaV2.1 channels unless the binding af-finity of CaV β GK domain is reduced by mutating the α-bindingpocket (ABP) site (28). Thus, in vitro work has emphasized highstability of CaV–β complexes whereas intact-cell work has em-phasized dynamic exchange of β subunits.Here, we developed chimeric translocatable CaV β subunits. They

can be translocated rapidly by chemically inducible heterodimerization

Significance

Voltage-gated Ca2+ (CaV) channels have an α1-α2δ core com-plexed with one of several alternative β subunits. Contradictoryevidence says that, once bound, (i) a β subunit is permanentlyassociated with the α1-α2δ core or (ii) that it is free to beexchanged for other β subunits. We designed rapamycin-translocatable CaV β subunits that allow drug-induced seques-tration of free β subunits to several organelle anchors. Seques-tering free subunits does not dissociate bound subunits fromchannels except when the binding site is mutated toweaken theinteraction. Nevertheless, our rapamycin constructs show that,when nontranslocatable β subunits are coexpressed with atranslocatable subunit, sequestering the translocatable subunitchanges the channel properties, revealing a quick replacementby the nontranslocatable subunit in the channel complex.

Author contributions: J.-H.Y., C.-G.P., B.H., and B.-C.S. designed research; J.-H.Y. andC.-G.P. performed research; J.-H.Y., C.-G.P., and B.-C.S. analyzed data; and J.-H.Y., B.H.,and B.-C.S. wrote the paper.

Reviewers: D.L., Brown University; and D.L.M., University of California, San Francisco.

The authors declare no conflict of interest.

Published under the PNAS license.1To whom correspondence may be addressed. Email: hille@uw.edu or bcSuh@DGIST.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1809762115/-/DCSupplemental.

Published online September 26, 2018.

E9934–E9943 | PNAS | vol. 115 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1809762115

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

to a membrane anchor on an intracellular organelle, includingthe plasma membrane, mitochondria, and endoplasmic reticu-lum (ER), upon addition of rapamycin. Using this method, rapidsequestration of free β subunits helped us to analyze the stabilityof CaV α1–β complexes in intact cells and the roles of β subunitsin channel gating. The heterodimerization is based on formationof a ternary complex between recombinant FK506 binding pro-tein (FKBP) and FKBP-rapamycin binding (FRB) protein uponaddition of rapamycin (29–31). In our experiments, chimeric βsubunit-FKBP proteins were heterodimerized through rapamy-cin with an FRB anchor on a target organelle. The designedtranslocatable CaV β systems permit study of association or dis-sociation of β subunits from CaV α1B channels located in theplasma membrane in living cells. We provide evidence that theinteraction of CaV α1 with β subunits can be very stable insidecells. The complex does not dissociate up to several hours.However, if there are other types of free β isoforms around thechannels, there can be exchange with the coupled β subunits onthe α1B protein. The CaV α1–β interaction can be weakened bymutating two residues in the α-binding pocket (ABP) site of the

CaV β GK domain (28). We show that acute dissociation of βsubunits from CaV α1B dramatically changes the functionalproperties of CaV2.2 channels.

ResultsGeneration and Characterization of Translocatable CaV β Subunits.CaV β subunits are noncovalently coupled to membrane-residentCaV α1 subunits of living cells (32). To further understand theinteraction properties, we used rapamycin-induced translocationof β subunits. CaV β isoforms were labeled at the C terminus withrecombinant FKBP and then in tandem with green (or mCherry)fluorescent protein; this produced the final β-FKBP-GFP (β-FG)proteins that can be recruited to specific subcellular target or-ganelles by rapamycin (Fig. 1A and SI Appendix, Fig. S1). Thetarget organelles were labeled with FRB and mCherry-fluorescentprotein fused to organelle membrane-specific marker proteins:Lyn11 for the plasma membrane (LDR), Tom20 for mitochondrialouter membranes (Tom20-MR), and Cb5 for the ER (MR-Cb5).FKBP and FRB construct pairs allowed rapid sequestration ofrecombinant 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. β-FG plasmids are constructed by fusing FKBP and EGFP to the C terminus of β subunit isoforms. Target organelle anchorsare constructed by labeling organelle-specific domains with FRB and mCherry (mCh). The organelle-specific domains are Lyn11, Tom20, and Cb5 for plasmamembrane (PM), mitochondria (Mito), and endoplasmic reticulum (ER), respectively. (B) Diagram of the rapamycin-induced PM translocatable β-FG system.The PM-targeted marker LDR has FRB anchored to the PM via the myristoylation and palmitoylation modification sequence Lyn11. After addition of rapa-mycin, β-FG is recruited to the PM from the cytosol by forming the tripartite complex FRB–rapamycin–FKBP. (C) Representative confocal fluorescence imagesof cells expressing LDR and β-FG without α1 subunits and line-scan analyses (Right) of β-FG before and after 100 nM rapamycin application for 2 min. The linescans are as follows: Control, white line in images and black line in analysis graph; +Rapa, orange lines. (Scale bars: 10 μm.) (D) Rapamycin (Rapa)-inducedchanges of cytosolic GFP fluorescence intensity in cells expressing LDR and translocatable β-FG subunits without CaV α1. Normalized cytosolic fluorescenceintensities are shown as a function of time. F/F0, fluorescence divided by initial fluorescence (n = 5). (E) Confocal fluorescence images of cells expressingmitochondrial anchor Tom20-MR and β-FG without α1 subunits before and after a 2-min application of rapamycin. Green fluorescence intensity in mito-chondria is measured before and after 2 min in rapamycin, and the GFP intensities (A.U., arbitrary unit) are presented in the Right (n = 5). *P < 0.05, comparedwith control. See SI Appendix, Fig. S2B for the ROIs selected for mitochondrial GFP intensity measurements in cells. (F) Confocal fluorescence images of cellsexpressing ER marker MR-Cb5 and β-FG without α1 before and after rapamycin addition. The selected ROIs are magnified on the Rightwhere the arrow headsindicate puncta formed by the β2a–FG complex with MR-Cb5 in the ER-PM junctions. See also SI Appendix, Fig. S2D for merged images. (G) β2a-FG and MR-Cb5 fluorescence intensities are measured at the PM before (Control) and after rapamycin addition. Note that the MR-Cb5 signal at the PM was weak incontrol cells but significantly increased and colocalized with β2a-FG (arrow heads) in puncta after rapamycin addition. (Scale bars: 10 μm.) All images arerepresentative of 7 to 10 cells in three independent experiments.

Yeon et al. PNAS | vol. 115 | no. 42 | E9935

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

of an FKBP–rapamycin–FRB complex (Fig. 1 A and B). We callthese FRB constructs anchors. In cells coexpressing β-FG iso-forms and the plasma membrane targeting anchor LDR, all of thecytosolic β-FG proteins translocated to the plasma membranewith time constants of ∼25 to 30 s after addition of 100 nMrapamycin (Fig. 1 C and D). Note that the β2a-FG isoform withtwo palmitoyl side chains in its N terminus was already well-localized to the plasma membrane in control cells and showedno change during addition of rapamycin. Similarly, in cellsexpressing the mitochondrial anchor Tom20-MR or the ER an-chor MR-Cb5, all cytosolic β-FG isoforms rapidly translocated tothe mitochondria or the ER, respectively, with rapamycin addition(Fig. 1 E and F and SI Appendix, Fig. S2). In cells expressing MR-Cb5, there was one difference: After rapamycin addition, theplasma membrane-localized palmitoylated β2a-FG congregated toform puncta at ER-plasma membrane (ER-PM) junctions thatbecame clearly visible in the confocal microscope within 2 min(Fig. 1F and SI Appendix, Fig. S2D). Line scans of the plasmamembrane revealed that β-FG and MR-Cb5 were colocalized inthe puncta (Fig. 1G). Presumably, transient ER-PM junctionsform often in the life of cells, and they are stabilized here whenrapamycin cross-links plasma membrane channels to an ER an-chor. Such control experiments validate translocatable β subunitsfor use in intact living cells.

Unlike Palmitoylated β2a-FG, the Electrostatically Tethered β2e-FGIsoform Could Translocate to Mitochondria. Like the β2a subunit,β2e localizes to the plasma membrane even without CaV α1 sub-units being present. It is not lipidated, but a polybasic region and ahydrophobic tryptophan residue in the β2e N terminus makeelectrostatic and hydrophobic interactions with acidic phospho-lipids in the plasma membrane (33–35) (SI Appendix, Fig. S3A).To probe this interaction, we used translocation in cells expressingβ2e-FG and mitochondrial Tom20-MR. Without α1 subunits, β2e-FG was primarily localized at the plasma membrane (SI Appendix,Fig. S3B). When the cells were treated with rapamycin, the fluo-rescence intensity of β2e-FG (green) decreased at the plasmamembrane and clearly increased in the mitochondrial region. Bycomparison, the fluorescence intensity of Tom20-MR (red) on themitochondria did not change. Evidently, unlike for palmitoylatedβ2a-FG, rapamycin almost completely removes β2e-FG from theplasma membrane, translocating it to the mitochondria within2 min. When β2e-FG was coexpressed with ER-localized MR-Cb5, the two proteins became colocalized in puncta at ER-PMjunctions after rapamycin addition (SI Appendix, Fig. S3C). Thesedata indicate that the electrostatic interaction of CaV β2e subunitswith the plasma membrane (i) is weak enough to be overcome byrapamycin-induced dimerization to distant and not very movablemitochondria but (ii) is not overcome when rapamycin links it tothe more compliant ER that can readily make PM junctions sta-bilized by other molecules and that apparently does not pull hardenough to tear β2e subunits off the PM.

The Phenotype of Interaction Between CaV α1B and β Subunits inCells. Next, we investigated the nature of CaV α1–β interactionsin live cells using confocal microscopy. When expressed alone intsA-201 cells, the β2a and β2e isoforms were mostly localized atthe plasma membrane whereas β1b, β2a(C3,4S), β2b, and β3isoforms were distributed through the cytosol with some in thenucleus (SI Appendix, Fig. S4A). However, when they wereexpressed together with CaV α1B/α2δ1 subunits, the β2a(C3,4S)and β2b isoforms were found in the plasma membrane only orboth in the plasma membrane and the cytoplasm in over 80% ofcells. This suggested that β2a(C3,4S) and β2b isoforms are as-sociated with α1B/α2δ1 complexes in the plasma membrane(SI Appendix, Fig. S4 A and B). However, in similar conditions,the β1b and β3 isoforms remained largely in the cytoplasm andnucleus. Their cytoplasmic distribution was not due to a higherexpression of β subunits compared with the α1B in the cells since,when the molar ratio of transfected β isoforms versus α1B wasdecreased by 50%, most β1b subunits still were located in the

cytoplasm and their overall subcellular distribution was notchanged (SI Appendix, Fig. S5 A and B). With β3 subunits, thecytosolic distribution actually increased at the lower ratio, per-haps because fewer α1B subunits were successfully trafficked tothe plasma membrane (SI Appendix, Fig. S5C). The expression ofα1B did not change the already high plasma membrane distri-bution of β2a and β2e isoforms.The isotype-selective expression patterns of β subunits also

contributed to a difference in electrophysiological properties ofCaV2.2 channels. Throughout, we used Ba2+ as the current carrier.SI Appendix, Fig. S4C compares representative current inactivationtraces of channels formed with different β-FG isoforms. Withplasma membrane-located β2a or β2e isoforms, the currents inac-tivated very slowly whereas, with cytosolic β isoforms, they inacti-vated rapidly (SI Appendix, Fig. S4D). In addition, we found thatthe average peak CaV2.2 current density of cells expressing chan-nels with plasma membrane-localized β isoforms was approximatelytwofold higher than for those with cytosolic β isoforms (SI Ap-pendix, Fig. S4E).

Interaction Between CaV α1B and β Subunits Is Stable in Cells. Is theCaV α1–β interaction stable or dynamic? We approached thisquestion with our translocation technique. In cells coexpressingthe mitochondrial anchor Tom20-MR, we first tested the effectsof rapamycin on CaV2.2 channels with β2 isoforms. As shown inFig. 2A, the palmitoylated β2a-FG, the palmitoylation-resistantmutant β2a(C3,4S), and nonpalmitoylated β2b all were mainlylocalized at the plasma membrane in the presence of CaV α1B/α2δ1. Rapamycin treatment for 2 min did not trigger any re-distribution of β2-FG isoforms from the plasma membrane tomitochondria. Fig. 2C shows peak current-voltage relations forthe different CaV2.2 channels in patch clamp electrophysiology.The vertical axis reflects the maximum current amplitude, andthe position on the voltage axis reflects the voltage dependenceof activation gating. With rapamycin addition, there were nochanges in the current amplitudes, in the activation curve (Fig. 2C and E), or in the time course of inactivation gating (SI Ap-pendix, Fig. S6 A and B). Apparently, CaV α1B–β coupling isquite stable on a 2-min time scale in these intact cells.We also investigated α1B–β binding stability in cells expressing

the ER anchor MR-Cb5. Consistent with Fig. 1 F and G, rapa-mycin formed puncta of membrane-localized β2a-FG and MR-Cb5 at ER-PM junctions (Fig. 2B). Results were similar for cellsexpressing CaV2.2 with β2a(C3,4S)-FG or β2b-FG. Nevertheless,formation of puncta did not result in changes in the currentamplitudes, in the activation curve, or in inactivation gating ofCaV2.2 currents (Fig. 2 D and E and SI Appendix, Fig. S6 C andD). Apparently, β-FG and CaV α1B/α2δ1 translocate togetherlaterally along the plasma membrane to the ER-PM junctionswithout change in the composition or gating properties of channelcomplexes. The diagram in Fig. 2F illustrates movement ofCaV2.2 channels complexed with β-FG to the ER-PM junctionsvia rapamycin-induced heterodimerization. Results were similarwith P/Q-type CaV2.1 and R-type CaV2.3 channels, but not withT-type CaV3 channels, which do not complex with β subunits (SIAppendix, Fig. S7).Unlike the β2 isoforms, β1b-FG and β3-FG were found both in

the cytoplasm and plasma membrane in cells expressing CaV α1B/α2δ1 (SI Appendix, Fig. S4B), consistent with previous reports (36–38). We examined whether some of those β subunits were interactingwith membrane α1B subunits. As shown in Fig. 3A, rapamycintreatment rapidly translocated the cytosolic β-FG subunits tomitochondria. After the translocation, significant residual greenfluorescence remained at the plasma membrane, implying thatsome β1b-FG and β3-FG subunits were stably associated with α1Bsubunits. Interestingly, the currents were not affected by the ad-dition of rapamycin (Fig. 3 B and C), suggesting that, once β1b orβ3 subunits were bound to α1B, they remained as intact com-plexes, still modulating channel gating. In cells cotransfected withthe ER anchor MR-Cb5, a significant number of puncta formed inER-PM junctional regions after rapamycin addition (Fig. 3D).

E9936 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

Still, the current density and activation curve were not affectedduring rapamycin-induced translocation to puncta (Fig. 3 E–G),indicating again that some β1b and β3 subunits remain firmly as-sociated with α1B even after they are pulled into ER-PM junctions.We further confirmed with two-color confocal microscopy that

α1B pore-forming subunits migrate to puncta together with thecoupled β-FG. We used α1B labeled with YFP in the C terminus(α1B-YFP), β1b-FKBP-mCherry (β1b-FmCh), and dark FRB-Cb5. Rapamycin treatment induced formation of puncta con-taining both α1B-YFP and β1b-FmCh colocalized at the plasmamembrane (Fig. 3 H and I). Similar behavior was found in cellscoexpressing β3-FmCh rather than β1b-FmCh (SI Appendix, Fig.S8). These observations suggest that some of the β1b and β3subunits interact stably with CaV α1B in intact cells, althoughmost of them are soluble in the cytosol, and that, with rapamycin,the full channel complex migrates to ER-PM junctions along theplasma membrane. The initial α1B–β interaction remained up to2 h after rapamycin as evidenced by no changes in the currentdensity and current inactivation (SI Appendix, Fig. S9). This setsan upper limit to the rate of dissociation and agrees well with invitro work that says these complexes are very stable.Can recruiting cytosolic β subunits to the plasma membrane

enhance CaV2.2 currents? We triggered translocation ofcytosol-localized β-FG isoforms to the plasma membrane withrapamycin (Fig. 3J). The translocation of β1b-FG and β3-FGisoforms was rapid (Fig. 3K). Nevertheless, the CaV2.2 currentsdid not change (Fig. 3 L and M), suggesting that all α1B sub-

units at the plasma membrane already were fully complexedwith β subunits.

A Dynamic Exchange of β Subunits Is Revealed When Two Different βIsoforms Are Expressed. In the experiments so far, we showed that,when translocatable free β subunits are targeted to an organelleanchor by the addition of rapamycin, the existing α1B–β channelcomplexes do not give up their bound β subunits. This apparentstability contrasts with literature reports of β subunit exchange incells where one kind of β subunit apparently can displace another.To imitate the reported conditions, we compared rapamycin ef-fects in cells coexpressing CaV2.2 and translocatable β2e-FG alonewith cells coexpressing CaV2.2 and both β2e-FG and additionalnontranslocatable cytosolic β1b or β3 in a 1:1 ratio. With two kindsof β subunits, we would expect a mixed population of channels,some containing the β2e-FG subunits and inactivating slowly andothers containing β1b or β3 subunits and inactivating rapidly as isseen in the black control traces in the middle and right columns ofFig. 4B. For comparison, the left column of the figure shows onlyslowly inactivating current when cells express only β2e-FG sub-units. As indicated diagrammatically in Fig. 4A, if one kind of βsubunit could displace another, then when the pool of trans-locatable β2e-FG is sequestered to mitochondria by rapamycin,the mixed β subunit population in the channel complexes mightgradually be replaced with 100% of the nontranslocatable β1b orβ3 subunits, and the characteristics of the recorded current would

Fig. 2. Stable interaction of β2 subunits with α1B in intact live tsA-201 cells. (A and B) Representative confocal fluorescence images of cells expressingCaV2.2 channels with different β2-FG isoforms and either Tom20-MR (A) or MR-Cb5 (B) before and after a 2-min application of rapamycin (100 nM). (Scalebars: 10 μm.) Puncta are found in cells with β2-FG isoforms and MR-Cb5 (B), and some of them are indicated by arrow heads in magnified merged images. (Cand D) Rapamycin effects on CaV2.2 channels in cells cotransfected with different β2-FG isoforms and Tom20-MR (C) or MR-Cb5 (D). Peak inward Ba2+ currentsare shown as a function of time. The currents are measured every 4 s by applying a depolarizing pulse to +10 mV. (Right) Normalized peak current-voltage (I-V) relations of currents evoked by voltage steps to the indicated potential. Relative inward currents are measured with voltage steps from −50 to +40 mV, in10-mV intervals, from a holding potential of −80 mV. Data are mean ± SEM of three independent experiments (n = 12 for all in Tom20-MR; n = 10 for all inMR-Cb5). (E) Summary of the rapamycin effects on CaV2.2 channels with different β2-FG isoforms with and without Tom20-MR (Left) or MR-Cb5 (Right). Dataare mean ± SEM. (F) Diagram of rapamycin-induced puncta formation at ER-PM junctions. Based on our results, rapamycin triggers puncta formation bytethering the ER membrane to the PM through complexes formed between α1B-bound β2-FG and ER-resident MR-Cb5 at the ER-PM junctional regions.

Yeon et al. PNAS | vol. 115 | no. 42 | E9937

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

eventually change to all fast inactivation. That indeed happened.In cells expressing CaV2.2 and β2e-FG alone, the slowly inacti-vating current changed little after rapamycin addition for 20 min(Fig. 4 B and C). However, in cells expressing β2e-FG togetherwith cytosolic β subunits, the proportion of CaV2.2 current thatinactivated rapidly increased significantly after addition of rapa-mycin (Fig. 4 B and C). This effect did not occur in the absence ofcoexpression of the Tom20 mitochondrial anchor. Similar butsmaller effects were seen in experiments where rapamycin wasapplied for only 10 min (SI Appendix, Fig. S10 A and B). Such datawould be consistent with the concept that cytosolic β1b orβ3 subunits are able to displace β2e subunits from existing channelcomplexes in <20 min.These observations merited more detailed examination. We

consider two alternative explanations: (i) could it be that slowlyinactivating channels just become inoperative after rapamycinaddition so thereby the relative fraction of rapidly inactivatingchannels appeared to rise in our normalized traces, or (ii) couldit be that newly inactivating channels are being inserted rapidly

through turnover, trafficking, or assembly and are diluting outthe existing population of slowly inactivating channels withoutthe supposed displacement of existing subunits? Since these ex-planations both postulate a change in the number of functionalchannels, we calculated the change of peak current for each cellafter 10 and 20 min of rapamycin (SI Appendix, Fig. S10C). Thesummarized results show that, as is common for recordings fromCaV2.2 channels, the currents run down in time; however, theaverage rate of decline does not depend on the expression ofeither Tom20 or cytosolic β subunits (SI Appendix, Fig. S10D).There is no evidence for incapacitation, turnover, or supplemen-tation of channels dependent on Tom20, rapamycin, or β subunitsubtype. We further tried to estimate the extent of change in theproportion of rapidly and slowly inactivating channels in our ex-periments. We took the 500-ms time courses of recorded currentsfor pure β1b and β2e-FG subunits (without rapamycin) and thenmathematically combined these two templates to mimic the cur-rent time courses for mixtures exposed to rapamycin. This wasdone both for the cells in the 10-min experiment (SI Appendix, Fig.

Fig. 3. Stable interaction of β1b and β3 isoforms with α1B in intact tsA-201 cells. (A) Representative confocal fluorescence images of cells expressingCaV2.2 channels with Tom20-MR and β1b-FG (Top) or β3-FG (Bottom) before and after a 2-min application of rapamycin (100 nM). Significant fluorescencesignals are found at the PM after rapamycin addition as indicated by arrowheads. (B) Rapamycin effects on CaV2.2 channels in cells cotransfected with β1b-FGor β3-FG. Inward Ba2+ currents are measured every 4 s by applying a depolarizing pulse to 10 mV. (C) Summary of rapamycin effects on CaV2.2 channelcurrents with β1b-FG or β3-FG. Data are mean ± SEM of three independent experiments (n = 10 for β1b-FG; n = 12 for β3-FG). (D) Representative confocalimages of cells expressing CaV2.2 channels with MR-Cb5 anchor and β1b-FG or β3-FG before and after rapamycin addition. The selected ROIs are magnifiedBelow. Arrowheads indicate puncta forming at ER-PM junctions after rapamycin addition. (E) Rapamycin effects on CaV2.2 channels with β1b-FG or β3-FG. (F)Summary of the rapamycin effects on CaV2.2 channel currents with β-FG and MR-Cb5. Data are mean ± SEM of two independent experiments (n = 10 for β1b-FG; n = 10 for β3-FG). (G) Ba2+ currents in cells expressing α1B/α2δ1 with MR-Cb5 and β1b-FG or β3-FG isoforms before and after rapamycin addition. TheCaV2.2 currents are measured in whole-cell configuration, where cells are depolarized to +10 mV from −80 mV for 10 ms. (H) Confocal images of cellsexpressing α1B-YFP/α2δ1 with β1b-FKBP-mCherry (β1b-FmCh) and ER anchor FRB-Cb5 before and after a 2-min application of rapamycin (100 nM). The selectedROIs are magnified on the Right where the puncta formed between α1B-YFP and β1b-FmCh in the ER-PM junctions are indicated by the arrow heads. (I)Fluorescence intensities of α1B-YFP or β1b-FmCh measured in the PM before and after rapamycin. Note that both α1B-YFP and β1b-FmCh signals are stronglyincreased in puncta after rapamycin addition. (J) Schematic diagram of the rapamycin-induced translocation of cytosolic β-FG to the PM. Rapamycin triggersformation of the complex between cytosolic β-FG and LDR at the PM. (K) Confocal fluorescence images of cells expressing CaV2.2 channels with β-FG and LDRbefore and after the 2-min application of rapamycin. (L) Rapamycin effects on CaV2.2 channel currents in cells cotransfected with LDR and β1b-FG or β3-FG.(M) Summary of the rapamycin effects on CaV2.2 channels with different β-FG isoforms and LDR. Data are mean ± SEM (n = 7 for all). (All scale bars: 10 μm.)

E9938 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

S11A) and for those in the 20-min experiment (SI Appendix, Fig.S11B). For the 10-min rapamycin experiment, the estimatedproportion of rapidly inactivating channels was ∼45% if theTom20 anchor was not expressed and 68% if the Tom20 wascoexpressed, and, for the 20-min experiment, the proportionswere ∼36 to 84%, respectively. Thus, the half-time of the switch inkinetics might be roughly 15 min. We suggest that this is too fastfor normal turnover and represents instead the time course of adisplacement reaction with β2e-FG subunits being displaced bycytosolic β1b subunits on existing channels.One unanticipated observation in Fig. 4 B and C and SI Ap-

pendix, Fig. S10 A and B requires mention. In the cells with twotypes of subunits but not expressing the Tom20 anchor, there wasa surprising slowing in overall inactivation kinetics after contin-uous exposure to rapamycin for 10 and 20 min. This change canbe seen as a kinetic mismatch in fitting the rapamycin curvesusing templates that are from cells not treated with rapamycin inSI Appendix, Fig. S11. We can only point out that, in every celltype, there are endogenous rapamycin targets, some with FKBPand others with FRB domains, so addition of rapamycin still haseffects on cells without overexpression of exogenous FRB do-mains. Thus, endogenous FKBP will become dimerized with theFRB domain of the tyrosine kinase mammalian target of rapa-mycin (mTOR) and the FKBP domain of β2e-FG subunits onchannels will be dimerized with mTOR as well. The conse-quences of these interactions would be beyond the scope ofthis paper.

Acute Dissociation of β Subunits from CaV2.2 Channel ComplexesChanges the Gating Properties. Next, we wanted to observe thefunctional effects of acute dissociation of β subunits from intactCaV channels. We weakened the interaction between the α-bindingpocket (ABP) of β with the α-interaction domain (AID) of α1 bymutation. Our double-mutant translocatable β subunits (hereafternamed “mutant β-FG”) had methionine (M) and leucine (L) res-idues of the ABP replaced by alanine (A). These mutations reducethe AID-ABP interaction affinity by ∼250-fold (23), allowingspontaneous dissociation of mutant β subunits from CaV2.1 channelcomplexes within 5 min in inside-out patches (28). The followingexperiments showed that sequestration by rapamycin addition candissociate these mutant β subunits from CaV2.2 channel complexes.The first experiment used only a membrane-localized I-II loop ofα1B (including the AID domain) rather than the full α1B loopsubunit. SI Appendix, Fig. S12 shows the subcellular localization ofmutant β-FG isoforms in cells coexpressing (i) the α1B I-II loopcoupled to Lyn11 and CFP (called Lyn-I-II loop-CFP) with (ii)Tom20-MR. In control, the mutant isoforms of β1, β2a(C3,4S), andβ2b were mainly distributed at the plasma membrane (presumablycoupled to Lyn-I-II loop-CFP) with some in the cytosol whereas themutant β2a forms were principally at the plasma membrane, pre-sumably through their palmitoylation. Interestingly, the mutantβ3 forms were detected only in the cytosol. When the cells weretreated with rapamycin, the mutant isoforms of β1b, β2a(C3,4S),β2b, and β3 translocated dramatically to the mitochondria from theplasma membrane, suggesting that the mutant subunits are readilydissociated from the AID domain and sequestered upon the ad-dition of rapamycin.We turned to the functional effects of acute β subunit dissoci-

ation from intact CaV2.2 channels using the Tom20-MR anchor.In accordance with weakened mutant β subunit binding, rapa-mycin addition significantly decreased inward currents throughCaV2.2 channels coupled with the mutated versions of β1b-FG,β2a(C3,4S)-FG, and β2b-FG subunits (Fig. 5 A and B). The effecttook 10 to 30 s. Rapamycin had little effect on the current ofchannels with the plasma membrane-bound mutant β2a-FG sub-unit, and it had little effect on channels with mutant β3-FG, whichwe suspect binds much more weakly to α subunits than the othermutant β subunits. After rapamycin application, the inactivationkinetics of Ba2+ currents in CaV2.2 channels with dissociablemutant β isoforms were almost as slow as for channels without anyβ subunits (No β) (Fig. 5C). Thus, the fraction of Ba2+ currentremaining after 100-ms depolarizations to +10 mV (r100) wasincreased with mutant isoforms of β1b-FG, β2a(C3,4S)-FG, andβ2b-FG (Fig. 5D). Interestingly, the inactivation of channels withmutant β3-FG was almost the same as the inactivation of channelswithout a β subunit. Upon rapamycin addition, we also found asignificant decrease in maximum current amplitudes in channelswith mutant versions of β1b-FG and β2b-FG (Fig. 5E). At thesame time, the voltage dependence of activation gating of thosechannels shifted to the right by up to ∼10 mV, approaching that ofchannels without β subunits (SI Appendix, Fig. S13). Here, thedependence of tail-current amplitude on prepulse voltage servesas a direct measure of the voltage dependence of activation gating.Before rapamycin addition, the tail current activation curve of

CaV2.2 channels in cells expressing the mutant β3-FG subunitwas almost the same as for channels without any β subunits (Noβ), and adding rapamycin had no effect. Both observations sug-gest that the steady-state fraction of CaV2.2 channels bound tomutant β3-FG subunits is quite small. Similarly, in confocal ex-periments where cells expressing Lyn-I-II-CFP with mutant β1b-FG and MR-Cb5 formed puncta at the plasma membrane afterrapamycin, the cells with mutant β3-FG did not (SI Appendix,Fig. S14). Nevertheless, we do see evidence of some α1B-mutant-β3 interaction since the mutant β3 still induced a small increasein the membrane expression of the α1B channel proteins com-pared with no-β cells (Fig. 5E). This increase was much less thanfor the other mutant subunits. We envision that, even if the steady-state binding fraction were, e.g., only 5 to 10%, the transientlybound complexes might be aided in trafficking to the surface before

Fig. 4. Dynamic interaction of β2e-FG with CaV α1B subunits in the presenceof other cytosolic β isoforms. (A) Diagram of experiment to test if the in-teraction of α1B–β subunits is dynamic in the presence of other cytoplasmic βisoforms. TsA-201 cells expressing CaV2.2 and rapamycin-translocatable β2e-FG alone or together with other cytosolic β subunits were treated with20 nM rapamycin for 20 min, and the Ba2+ currents before and after rapa-mycin were measured. FRB is anchored to the mitochondrial (Mito) outermembrane via mitochondrial import receptor subunit Tom20 (Tom20-MR).(B) Representative Ba2+ current traces showing inactivation gating in cellsexpressing α1B/α2δ1 and β2e-FG alone or with β1b or β3 isoforms before(Control) and 20 min after addition of rapamycin (20 nM). The CaV2.2 cur-rents are measured in whole-cell configuration in cells depolarized to+10 mV from −80 mV for 500 ms. (C) Summary of the rapamycin effects onCaV2.2 current inactivation with β2e-FG alone or with other β isoforms. Thefraction of Ba2+ current remaining after 100-ms depolarization to +10 mV(r100) is compared before (Control) and after rapamycin addition. Data aremean ± SEM of three independent experiments (n = 5–7). *P < 0.05, **P <0.01, compared with control.

Yeon et al. PNAS | vol. 115 | no. 42 | E9939

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

the mutant β3 subunit dissociated again. The same effects of thevarious mutant β-FG subunits were also detected in experimentswith CaV2.3 channels. Thus, when the mutant β1b-FG was acutelyrecruited away from CaV2.3 channels, current amplitudes fell, andthe tail current activation curve was shifted to the right by ∼9 mV(SI Appendix, Fig. S15). Collectively, we find that, when the βsubunit is acutely dissociated from CaV α1B, the current densitydecreases, the inactivation gating slows, and the activation curveshifts to more positive potentials.

Dissociation of β Subunits from α1B Decreases the Phosphatidylinositol4,5-Bisphosphate Sensitivity of CaV2.2 Channels. Plasma membranephosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] can be depletedrapidly by the voltage-sensing phosphatase Dr-VSP, a poly-phosphoinositide phosphatase enzyme activated by depolarizationof the membrane potential (39, 40). We used Dr-VSP to comparethe PI(4,5)P2 sensitivity of channels without and with various CaV-βsubunits. Without any β subunits, CaV α1B/α2δ1 channels were onlyweakly (∼10%) inhibited by PI(4,5)P2 depletion, similar to the re-sponses of CaV channels with the membrane-targeted β2a subunits(Fig. 6 A and B). Nevertheless, inactivation gating of the β-lesschannels was relatively fast and more similar to that of channelswith cytosolic β subunits (SI Appendix, Fig. S16).We measured the PI(4,5)P2 sensitivity of CaV2.2 channels with

translocatable mutant β isoforms before and after rapamycinaddition. As shown in Fig. 6C, currents in channels with unmu-tated WT cytosolic β1b, β2b, or β3 subunits were significantlyinhibited by PI(4,5)P2 depletion, and this current inhibition wasnot affected by rapamycin addition. However, with loosely boundmutant cytoplasmic β1b or β2b subunits, rapamycin reduced theinhibition by PI(4,5)P2 depletion. Channels with membrane-tethered WT or mutant β2a were insensitive to depletion of PI(4,5)P2 (Fig. 6 C and D). Finally, whereas channels with WT

β3 subunits were strongly inhibited by PI(4,5)P2 depletion, thechannels with mutant β3-FG showed only slight inhibition by PI(4,5)P2 depletion and no effects of rapamycin addition. This wasconsistent with our suggestion that very few CaV α1B/α2δ1channels are bound to mutant β3 subunits at any moment so theybehave like channels without β subunits (No β) (Fig. 6 C and D).Thus, bound β subunits are needed for PI(4,5)P2 regulation ofCaV channels, and their subcellular location is critical for thedetermination of PI(4,5)P2 sensitivity.

DiscussionPublished work with recombinant expression systems shows thatCaV β subunits both increase cell surface expression of α1 subunitsand modulate the biophysical properties of HVA CaV channelgating (2–4). Here, we developed chemically translocatable (30, 31)CaV β subunits to examine interactions between α1B and β subunitsin CaV2.2 channel complexes and their real-time regulatory effectsin intact cells. We initially used the dimerization system to sequesterall free subunits to the plasma membrane, ER, or mitochondria.Those results showed that the in-cell α1B–β interaction can lookvery stable for some of the β isotypes, especially those that are in-trinsically membrane-localized. However, coexpressing additionalfunctionally different free β isoforms revealed that one β subunit canbe exchanged for another, changing properties of the channel suchas modulation by membrane depolarization and PI(4,5)P2 metabo-lism. We review that evidence and at the end develop a hypothesisthat reconciles these apparently contradictory observations. In ad-dition, we showed that acute removal of β subunits reduces currentsby up to 50% in less than 1 min and changes the gating kinetics.

Subcellular Distribution of β Subunits. Consider first the localiza-tion of β subunits in the absence of any α1 subunit. The β1b andβ3 subunits are found in the cytosol, since they lack binding

Fig. 5. Reversible interaction of mutant β-FG iso-forms with CaV α1B subunit in intact tsA-201 cells.The double-mutant subunits are abbreviated (MA,LA), short for M248A and L252A mutations. (A) Timecourse of rapamycin effects on CaV2.2 peak currentsin cells coexpressing diverse mutant β-FG isoformswith or without Tom20-MR. Inward Ba2+ currents aremeasured every 4 s by applying a depolarizing pulseto +10 mV. (B) Summary of the rapamycin effects onCaV2.2 currents with mutant β-FG isoforms. Data aremean ± SEM of three independent experiments [n =10 for β1b-FG; n = 8 for β2a-FG; n = 10 for β2a(C3,4S)-FG; n = 10 for β2b-FG; n = 12 for β3-FG]. ***P < 0.001,compared with –Tom20. (C ) Representative Ba2+

current traces showing inactivation gating in cellsexpressing α1B/α2δ1 without (No β) or with variousmutant β-FG isoforms and Tom20-MR before (Con-trol) and after rapamycin addition. The CaV2.2 cur-rents are measured in whole-cell configurationwhere cells are depolarized to +10 mV from −80 mVfor 500 ms. (D) Summary of the rapamycin effects oncurrent inactivation of CaV2.2 channels with mutantβ-FG isoforms and Tom20-MR. The fraction of Ba2+

current remaining after 100-ms depolarization to+10 mV (r100) is compared before (Control) and af-ter rapamycin addition. Data are mean ± SEM, *P <0.05, **P < 0.01, compared with control. (E) Peakcurrent-voltage (I-V) relations of Ba2+ currentsevoked by voltage steps to the indicated potential(mV) in cells expressing CaV2.2 channels without (Noβ) or with mutant β-FG isoform and Tom20-MR. In-ward current density is measured with voltage stepsfrom −50 to +40 mV, in 10-mV intervals, from aholding potential of −80 mV. Data are mean ± SEM.

E9940 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

partners at the plasma membrane, whereas β2a and β2e subunitsare tethered to the plasma membrane through two palmitoyl sidechains or nonselective electrostatic interactions, respectively(33–35, 41). When α1 subunits are coexpressed with WT β sub-units, all β subtypes showed partial or total membrane localiza-tion, consistent with previous experiments in native cells andrecombinant systems (36–38). We could demonstrate significantassociation of the β1b and β3 subunits with α1B subunits at theplasma membrane since, when these membrane-localized βsubunits were translocated and concentrated in punctate ER-PMjunctions by rapamycin application, the α1B subunits went withthem. Translocating the cytosolic free β1b or β3 subunits to theplasma membrane upon addition of rapamycin did not increaseCaV currents as if all of the α1B subunits at the plasma mem-brane were already functionally partnered with a β subunit.Additional functional roles of the free cytosolic β1b and β3 iso-forms are not clear, but it has been suggested that they could playregulatory roles by interacting with other cytosolic proteins (42).Thus, a recent report showed that cytosolic β3 subunits reduceinositol 1,4,5-trisphosphate (IP3) formation and receptor-inducedCa2+ release by physically forming β3/IP3 receptor complexesthrough the SH3-HOOK domain (43).

Real-Time Effects of β Subunit Dissociation on CaV2.2 Channel Gating.Previous studies of α1 and β subunit interactions in Xenopusoocytes reported that selective mutations in the AID and ABPinteraction sites weaken the interaction and decrease delivery ofchannel complexes to the plasma membrane (21, 23). The in-teraction could be weakened by mutating two residues, Met andLeu, in the ABP site of the β subunit GK domain (28). In ourhands, such mutated β subunits have a lower affinity for the I-IIloop of α1B subunits and are easily dissociated from the channelcomplex by rapamycin addition. The rapamycin-induced disso-

ciation was not always the same. It was greatest in channels withmutant β1b subunits, where rapamycin decreased the currents by∼55%, and relatively less in channels with mutant β2 subunits.Interestingly, rapamycin did not change gating of channelswith mutant β3 subunits at all as if there is only infrequentinteraction between α1B and mutant β3 subunits. These resultsagree with earlier work showing that β subunits interact withα1 through both the high-affinity AID region and a lower af-finity C terminus and that the interaction through the loweraffinity binding site is isotype-specific in the order β2 >β1b >>> β3 (13, 14). Similarly, we found that, when the in-teraction between AID and ABP was weakened by doublemutations, the rapamycin effect on current inhibition wasgreater in channels with mutant β1b than mutant β2. We alsosuggest that the mutant β3 form interacts infrequently withα1B subunits so most mutant β3 subunits are located in thecytosol and the current is small and not affected by rapamycinaddition. Rapamycin had little effect on the gating ofCaV2.2 channels with mutant β2a subunits, including currentamplitude, voltage dependence of activation, and inactivationgating. Presumably, the palmitoyl side chains prevent thedissociation of mutant β2a from CaV α1B on the additionof rapamycin.Our results established the real-time effects of β subunit dis-

sociation from CaV2.2 channels on gating properties in live cells.(i) We found that the current amplitude fell sharply, consistentwith previous studies with mutant β subunits in inside-out patches,where the current decreased by 50% (28). (ii) We found that theinactivation of CaV2.2 channels with mutant β subunits was slowedand more similar to the inactivating current of CaV2.2 without βsubunits. At the same time, the voltage dependence of activationgating was shifted to the right and became less steep. We foundsimilar changes in in CaV2.3 channels.

Fig. 6. Modulation of CaV2.2 channels with mutant β-FG isoforms by Dr-VSP–induced PI(4,5)P2 depletion. (A, Left) Diagram showing Dr-VSP activated bymembrane depolarization through voltage-sensing domain (VSD) dephosphorylating PI(4,5)P2 at the 5 position via its cytosolic phosphatase domain (PD). (A,Right) Inhibition of CaV2.2 currents by Dr-VSP activation in tsA-201 cells coexpressing α1B/α2δ1 without (No β) or with β2a or β2b isoforms. The cells aredepolarized to +120 mV for 1 s to activate Dr-VSP. Currents are measured during test pulses before and after the strong depolarizing pulse in control (blacktraces) and cells expressing Dr-VSP (red traces). (B) Summary of CaV2.2 inhibition by membrane depolarization in control and cells expressing Dr-VSP. Data aremean ± SEM (n = 6 to 7 for control, n = 10 for Dr-VSP). NS, not significant. ***P < 0.001, compared with CaV2.2 channels without β subunits (No β). (C) Effectsof Dr-VSP–mediated PI(4,5)P2 depletion on CaV2.2 channels with diverse control and mutant β-FG isoforms before (Left) and after (Right) rapamycin addition.CaV2.2 currents before (a) and after the activation of Dr-VSP (b) are superimposed. Black traces are the control, and red traces are currents after Dr-VSPactivation. Peak tail currents are indicated by arrow heads. (D) Summary of Dr-VSP–induced current inhibition before and after rapamycin addition. The openbar shows the current inhibition by Dr-VSP in cells expressing CaV2.2 channels without β subunits (No β). Data are mean ± SEM (n = 8–12). Analysis is per-formed by one-way ANOVA followed by Dunnett’s post hoc test. **P < 0.01, ***P < 0.001, compared with current inhibition in control.

Yeon et al. PNAS | vol. 115 | no. 42 | E9941

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

Significance of β Subunits for CaV2.2 Channel Regulation by PI(4,5)P2.Plasma membrane PI(4,5)P2 is required for full activation ofHVA CaV channels, and, when PI(4,5)P2 is depleted by stimu-lation of Gq-coupled receptors or by externally expressed phos-phatases, the CaV currents fall (44, 45). We previously found thatthe PI(4,5)P2 sensitivity of HVA CaV channels is tightly con-trolled by the subcellular localization of CaV β subunits (37, 46).Thus, channels with plasma membrane-localized β subunits (β2aor β2e) are only weakly (∼10%) inhibited by PI(4,5)P2 depletionwhereas channels with cytosolic β subunits are strongly (30 to60%) inhibited (47). Here, we find that the intrinsic PI(4,5)P2sensitivity of β-less CaV2.2 channels is very weak and resemblesthat for channels with membrane-tethered β subunits. Theseresults invite speculation that the subcellular positioning offlexible I-II domains of β-less α1B subunits is similar to that withmembrane-targeted β subunits and that the soluble β subunitsaugment PI(4,5)P2 regulation of CaV2.2 channels by pulling theI-II loop of the α1B subunit in the cytosolic direction (48).

Interaction Properties of α1B–β Subunits in Intact Cells and a ClosingHypothesis.Our rapamycin-translocatable CaV β subunits are idealfor examining the stability of CaV α1–β interactions in intact cells.Using this system, we confirmed that, when a single type of βisoform is expressed, all WT α1B–β interactions are so strong thatthey are not broken by sequestering most free β subunits from thecytosol. In the presence of an ER anchor, the PM complexes areclustered to ER-PM junctions without changes in channel elec-trophysiology, and the currents are not changed for up to 2 h afterrapamycin addition. Such results would imply that the binding ofCaV β subunits to α1B is almost irreversible, a conclusion con-sistent with many previous papers showing that β2a does notdissociate from CaV α1A during inside-out patch clamp (28). Inaddition, CaV α1S/β1a and α1C/β1a do not dissociate in fluores-cence recovery after photobleaching (FRAP) experiments inskeletal muscle (26), and anti-β3 antibodies do not dissociate CaVα1A/β3 and α1B/β3 complexes in rat brain (49). However, theresults were different when we expressed two types of β subunittogether, one membrane tethered and rapamycin translocatableand the other primarily cytosolic and not rapamycin trans-locatable. Upon addition of rapamycin, the gating of channelswith mostly β2e subunits was changed to be like that of channelswith mostly cytosolic β subunits, demonstrating a removal oftranslocatable β2e subunits from the channels in exchange for theother soluble β subunits. We estimate an ∼15-min half-life for theexchange. These results are consistent with previous observationsof functional exchange of different β subunits in living cells (24–27, 50, 51). Apparently, the α1B–β interaction is dynamic in acompetitive binding situation.As a hypothesis, we propose that dissociation of a WT β

subunit from α1B is a concerted reaction requiring a second βsubunit to displace it. It will not happen when there are nocompeting subunits. Possibly, there are two parts to the in-teraction site: If a bound subunit transiently releases from onepart, a competing subunit might capture the revealed partial site,gaining a toehold to displace the bound subunit from the secondpart. Presumably, it is not necessary for the competing subunit tobe a different isoform, but, for our experiments, it is only whenthe displacement changes the gating properties of the channelthat our assay could document dissociation of one β subunit inexchange for another. In most cells, more than two β isoformsare expressed at the same time, and thus they would competewith each other continuously. Relative expression levels of the βisoforms might determine the subunit composition in CaVchannels and could contribute to dynamic modulation of thechannels in a particular neuron.

Materials and MethodsPlasmids, Cloning, and Mutagenesis. We used the following plasmids: ratCaV1.2 and rat β2a (from William A. Catterall, University of Washington,Seattle), rat β2a(C3,4S) and β2b (from Joyce Hurley, Indiana University,Indianapolis), rat CaV2.2e[37b] (52) and rat α2δ and rat β3 (from Diane

Lipscombe, Brown University, Providence, RI), CaV2.3 (from Terrance P.Snutch, University of British Columbia, Vancouver), Dr-VSP without EGFP(from B.H.), M2-muscarinic receptor (from Guthrie Resource Center), thechemical translocation systems Lyn11-FRB (LDR), Tom20-mCherry-FRB(Tom20-MR), and mCherry-FRB-Cb5 (MR-Cb5) (from Tobias Meyer, Stan-ford University, Stanford, CA), and CaV2.2-YFP (from David T. Yue, JohnsHopkins University, Baltimore). Rat β1b subunit was cloned from rat braincDNA. To generate translocatable CaV β-fluorescent protein fusions, FKBP wasamplified using PCR and subcloned into pEGFP-N1 or mCherry-N1 vectors(Clontech) using EcoRI and BamHI sites (SI Appendix, Fig. S1). To create β-FKBP-fluorescent proteins, β isoforms were amplified using PCR and subcloned intopFKBP-EGFP-N1 vector using multicloning sites NheI and EcoRI for β1b, β2a,β2a(C3,4S), and β2b and HindIII and EcoRI for β3. Point mutation of ABP inCaV β GK domain was achieved using the standard inverse PCR method. Primersfor plasmids in this study are listed in SI Appendix, Table S1.

Cell Culture and Transfection. TsA-201 cells were cultured in 35-mm culturedishes under standard growth conditions (37 °C and 5% CO2) in DMEM(Sigma-Aldrich) supplemented with 10% FCS (HyClone; Thermo Scientific)and 0.2% penicillin/streptomycin (HyClone; Thermo Scientific) at 37 °C under5% CO2. For the expression of CaV channel subunits, cells were transientlycotransfected with various subunits using Lipofectamine 2000 (Invitrogen)according to the manufacturer’s protocol. Unless stated, the transfectedDNA mixture consisted of plasmids encoding α1, β, and α2δ1 at a 1:1:1 molarratio. For electrophysiological recordings and confocal microscopy experi-ments, cells were plated onto poly-L-lysine–coated coverslip chips 24 to 36 hafter transfection and used within 12 to 24 h after plating.

Electrophysiological Recordings. Whole-cell patch-clamp recordings were ac-quired as described (47). Ba2+ currents were recorded in whole-cell config-urations using a HEKA EPC-10 patch-clamp amplifier with PatchMastersoftware (HEKA Elektronik). Some analysis used Fit Master software. Pipetteswere pulled from glass micropipette capillaries (World Precision Instruments)using a Flaming/Brown micropipette puller model P-97 (Sutter InstrumentCo.) and had resistances of 1.5 to 3.5 megohms when filled with internalsolution. Series-resistance errors were compensated at >60%, and fast andslow capacitance was compensated for before the applied test pulse se-quences. Currents were sampled at 10 kHz and filtered at 3 kHz. For allrecordings, cells were held at −80 mV. All recordings were leak- andcapacitance-subtracted before analysis by use of a –P/5 protocol. The ex-ternal Ringer’s solution contained 150 mM NaCl, 10 mM BaCl2, 1 mM MgCl2,10 mM Hepes, and 8 mM glucose; pH was adjusted to 7.4 with NaOH andosmolarity of 321 to 350 mOsm. The internal pipette solution consisted of175 mM CsCl, 5 mM MgCl2, 5 mM Hepes, 0.1 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 3 mM Na2ATP, and 0.1 mMNa3GTP; the pH was adjusted to 7.4 with CsOH and osmolarity of 321 to350 mOsm. All experiments were carried out at room temperature (20 to24 °C). The reagents were as follows: CsOH, BAPTA, Na2ATP, and Na3GTP(Sigma-Aldrich), and other chemicals (Merck).

Confocal Imaging. All imaging was performed with a Carl Zeiss LSM 700confocal microscope. For live-cell imaging, a 40× (water) apochromatic ob-jective lens at 1,024 × 1,024 pixels with digital zoom was used. For timecourses, 524 × 524 pixels were used. To analyze the time course of cytosolicfluorescence of various proteins, images were taken every 5 s in Zeiss ZEN im-aging software. Membrane localization was assessed from the line intensityhistograms of plasma membrane in the images after acquisition. Line-scan trackswere chosen to avoid the nucleus. Regions of interest (ROIs) were selected in thecytosolic or organelle regions of cells, and quantitative analysis was performedusing the “profile“ and “measure“ tools in ZEN 2012 lite imaging software (CarlZeiss MicroImaging). All confocal images were transferred in TIFF format, andraw data from the time course were processed with Microsoft Office Excel2012 and summarized in Igor Pro (WaveMetrics Inc.).

Statistical Analysis. Patch-clamp and imaging data were analyzed using IgorPro-6.37 (WaveMetrics), Excel 2016 (Microsoft), or ZEN 2012 (Carl Zeiss)software programs. Statistics in text or figures represent mean ± SEM. Sta-tistical comparisons were made by one-way or two-way ANOVA dependingon the number of experimental groups, followed by Tukey’s post hoc test.Differences were considered significant at the *P < 0.05, **P < 0.01, and***P < 0.001.

ACKNOWLEDGMENTS. We thank Dr. William A. Catterall for reading andhelpful discussion; and Lea M. Miller for technical assistance. We also thankmany laboratories for providing the plasmids. This work was supported by

E9942 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0

the following grants: a National Research Foundation of Korea (NRF) grantfunded by the Korea government [Ministry of Science, Information andCommunications Technology (ICT), and Future Planning] (2016R1A2B4014253)(to B.-C.S.), Basic Science Research Program Grant 2017R1A4A1015534 (toB.-C.S.), the Daegu Gyeongbuk Institute of Science and Technology R&D

Program of the Ministry of Science, ICT and Future Planning (18-BD-06) (toB.-C.S.), the Korea Brain Research Institute (KBRI) basic research programfunded by the Ministry of Science, ICT and Future Planning (18-BR-04-1) (toB.-C.S.), NIH Grant R37-NS08174 (to B.H.), and the Wayne E. Crill EndowedProfessorship (to B.H.).

1. Catterall WA (2011) Voltage-gated calcium channels. Cold Spring Harb Perspect Biol 3:a003947.

2. Dalton S, Takahashi SX, Miriyala J, Colecraft HM (2005) A single CaVβ can reconstituteboth trafficking and macroscopic conductance of voltage-dependent calcium chan-nels. J Physiol 567:757–769.

3. Buraei Z, Yang J (2010) The ß subunit of voltage-gated Ca2+ channels. Physiol Rev 90:1461–1506.

4. Dolphin AC (2012) Calcium channel auxiliary α2δ and β subunits: Trafficking and onestep beyond. Nat Rev Neurosci 13:542–555.

5. Ruth P, et al. (1989) Primary structure of the β subunit of the DHP-sensitive calciumchannel from skeletal muscle. Science 245:1115–1118.

6. Hullin R, et al. (1992) Calcium channel β subunit heterogeneity: Functional expressionof cloned cDNA from heart, aorta and brain. EMBO J 11:885–890.

7. Castellano A, Wei X, Birnbaumer L, Perez-Reyes E (1993) Cloning and expression of aneuronal calcium channel β subunit. J Biol Chem 268:12359–12366.

8. Pragnell M, et al. (1994) Calcium channel β-subunit binds to a conserved motif in the I-II cytoplasmic linker of the α1-subunit. Nature 368:67–70.

9. Witcher DR, De Waard M, Liu H, Pragnell M, Campbell KP (1995) Association of nativeCa2+ channel β subunits with the α1 subunit interaction domain. J Biol Chem 270:18088–18093.

10. Chen YH, et al. (2004) Structural basis of the α1-β subunit interaction of voltage-gatedCa2+ channels. Nature 429:675–680.

11. Van Petegem F, Clark KA, Chatelain FC, Minor DL, Jr (2004) Structure of a complexbetween a voltage-gated calcium channel β-subunit and an α-subunit domain. Nature429:671–675.

12. Tareilus E, et al. (1997) A Xenopus oocyte β subunit: Evidence for a role in the as-sembly/expression of voltage-gated calcium channels that is separate from its role as aregulatory subunit. Proc Natl Acad Sci USA 94:1703–1708.

13. Walker D, Bichet D, Campbell KP, De Waard M (1998) A β4 isoform-specific interactionsite in the carboxyl-terminal region of the voltage-dependent Ca2+ channel α1Asubunit. J Biol Chem 273:2361–2367.

14. Walker D, et al. (1999) A new β subtype-specific interaction in α1A subunit controlsP/Q-type Ca2+ channel activation. J Biol Chem 274:12383–12390.

15. DeWaard M, Witcher DR, Pragnell M, Liu H, Campbell KP (1995) Properties of the α1-βanchoring site in voltage-dependent Ca2+ channels. J Biol Chem 270:12056–12064.

16. Scott VE, et al. (1996) β subunit heterogeneity in N-type Ca2+ channels. J Biol Chem271:3207–3212.

17. Bell DC, et al. (2001) Biophysical properties, pharmacology, and modulation of human,neuronal L-type α1D, CaV1.3) voltage-dependent calcium currents. J Neurophysiol 85:816–827.

18. Cantí C, et al. (2001) Evidence for two concentration-dependent processes forβ-subunit effects on α1B calcium channels. Biophys J 81:1439–1451.

19. Geib S, et al. (2002) Use of a purified and functional recombinant calcium-channelβ4 subunit in surface-plasmon resonance studies. Biochem J 364:285–292.

20. Opatowsky Y, Chomsky-Hecht O, Kang MG, Campbell KP, Hirsch JA (2003) Thevoltage-dependent calcium channel β subunit contains two stable interacting do-mains. J Biol Chem 278:52323–52332.

21. Butcher AJ, Leroy J, Richards MW, Pratt WS, Dolphin AC (2006) The importance ofoccupancy rather than affinity of CaVβ subunits for the calcium channel I-II linker inrelation to calcium channel function. J Physiol 574:387–398.

22. Richards MW, Leroy J, Pratt WS, Dolphin AC (2007) The HOOK-domain between theSH3 and the GK domains of CaVβ subunits contains key determinants controllingcalcium channel inactivation. Channels (Austin) 1:92–101.

23. Van Petegem F, Duderstadt KE, Clark KA, Wang M, Minor DL, Jr (2008) Alanine-scanning mutagenesis defines a conserved energetic hotspot in the CaVα1 AID-CaVβinteraction site that is critical for channel modulation. Structure 16:280–294.

24. Hidalgo P, Gonzalez-Gutierrez G, Garcia-Olivares J, Neely A (2006) The α1-β-subunitinteraction that modulates calcium channel activity is reversible and requires acompetent α-interaction domain. J Biol Chem 281:24104–24110.

25. Obermair GJ, Tuluc P, Flucher BE (2008) Auxiliary Ca2+ channel subunits: Lessonslearned from muscle. Curr Opin Pharmacol 8:311–318.

26. Campiglio M, Di Biase V, Tuluc P, Flucher BE (2013) Stable incorporation versus dy-namic exchange of β subunits in a native Ca2+ channel complex. J Cell Sci 126:2092–2101.

27. Findeisen F, et al. (2017) Stapled voltage-gated calcium channel (CaV) α-Interactiondomain (AID) peptides act as selective protein-protein interaction inhibitors of CaVfunction. ACS Chem Neurosci 8:1313–1326.

28. Zhang Y, et al. (2008) Origin of the voltage dependence of G-protein regulation of P/Q-type Ca2+ channels. J Neurosci 28:14176–14188.

29. Banaszynski LA, Liu CW, Wandless TJ (2005) Characterization of the FKBP·rapamycin·FRBternary complex. J Am Chem Soc 127:4715–4721.

30. Inoue T, Heo WD, Grimley JS, Wandless TJ, Meyer T (2005) An inducible translocationstrategy to rapidly activate and inhibit small GTPase signaling pathways. Nat Methods2:415–418.

31. Suh BC, Inoue T, Meyer T, Hille B (2006) Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science 314:1454–1457.

32. Curtis BM, Catterall WA (1984) Purification of the calcium antagonist receptor of thevoltage-sensitive calcium channel from skeletal muscle transverse tubules. Biochemistry23:2113–2118.

33. Kim DI, Park Y, Jang DJ, Suh BC (2015) Dynamic phospholipid interaction of β2esubunit regulates the gating of voltage-gated Ca2+ channels. J Gen Physiol 145:529–541.

34. Kim DI, et al. (2015) Molecular basis of the membrane interaction of the β2e subunitof voltage-gated Ca2+ channels. Biophys J 109:922–935.

35. Kim DI, Kweon HJ, Park Y, Jang DJ, Suh BC (2016) Ca2+ controls gating of voltage-gated calcium channels by releasing the β2e subunit from the plasma membrane. SciSignal 9:ra67.

36. Xie M, et al. (2007) Facilitation versus depression in cultured hippocampal neuronsdetermined by targeting of Ca2+ channel CaVβ4 versus CaVβ2 subunits to synapticterminals. J Cell Biol 178:489–502.

37. Suh BC, Kim DI, Falkenburger BH, Hille B (2012) Membrane-localized β-subunits alterthe PIP2 regulation of high-voltage activated Ca2+ channels. Proc Natl Acad Sci USA109:3161–3166.

38. Mustafá ER, et al. (2017) Constitutive activity of the ghrelin receptor reduces surfaceexpression of voltage-gated Ca2+ channels in a CaVβ-dependent manner. J Cell Sci130:3907–3917.

39. Murata Y, Iwasaki H, Sasaki M, Inaba K, Okamura Y (2005) Phosphoinositide phos-phatase activity coupled to an intrinsic voltage sensor. Nature 435:1239–1243.

40. Keum D, Kruse M, Kim DI, Hille B, Suh BC (2016) Phosphoinositide 5- and 3-phos-phatase activities of a voltage-sensing phosphatase in living cells show identicalvoltage dependence. Proc Natl Acad Sci USA 113:E3686–E3695.

41. Chien AJ, et al. (1995) Roles of a membrane-localized β subunit in the formation andtargeting of functional L-type Ca2+ channels. J Biol Chem 270:30036–30044.

42. Berggren PO, et al. (2004) Removal of Ca2+ channel β3 subunit enhances Ca2+ oscil-lation frequency and insulin exocytosis. Cell 119:273–284.

43. Belkacemi A, et al. (2018) IP3 receptor-dependent cytoplasmic Ca2+ signals are tightlycontrolled by CaVβ3. Cell Rep 22:1339–1349.

44. Suh BC, Leal K, Hille B (2010) Modulation of high-voltage activated Ca2+ channels bymembrane phosphatidylinositol 4,5-bisphosphate. Neuron 67:224–238.

45. Jeong JY, Kweon HJ, Suh BC (2016) Dual regulation of R-type CaV2.3 channels by M1

muscarinic receptors. Mol Cells 39:322–329.46. Park CG, Suh BC (2018) Modulation mechanisms of voltage-gated calcium channels.

Curr Opin Physiol 2:77–83.47. Keum D, Baek C, Kim DI, Kweon HJ, Suh BC (2014) Voltage-dependent regulation of

CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β sub-unit. J Gen Physiol 144:297–309.

48. Wu J, et al. (2016) Structure of the voltage-gated calcium channel CaV1.1 at 3.6 Åresolution. Nature 537:191–196.

49. Bichet D, Lecomte C, Sabatier JM, Felix R, De Waard M (2000) Reversibility of the Ca2+

channel α1-β subunit interaction. Biochem Biophys Res Commun 277:729–735.50. Yamaguchi H, et al. (1998) Multiple modulation pathways of calcium channel activity

by a β subunit. Direct evidence of β subunit participation in membrane trafficking ofthe α1C subunit. J Biol Chem 273:19348–19356.

51. García R, Carrillo E, Rebolledo S, García MC, Sánchez JA (2002) The β1a subunit reg-ulates the functional properties of adult frog and mouse L-type Ca2+ channels ofskeletal muscle. J Physiol 545:407–419.

52. Lipscombe D, Pan JQ, Gray AC (2002) Functional diversity in neuronal voltage-gatedcalcium channels by alternative splicing of Cavα1. Mol Neurobiol 26:21–44.

Yeon et al. PNAS | vol. 115 | no. 42 | E9943

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Nov

embe

r 15

, 202

0