+ 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 27, 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. β-FG plasmids are constructed by fusing FKBP and EGFP to the C terminus of β subunit isoforms. Target organelle anchors

are constructed by labeling organelle-specific domains with FRB and mCherry (mCh). The organelle-specific domains are Lyn11, Tom20, and Cb5 for plasma membrane (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 images of cells expressing LDR and β-FG without α1 subunits and line-scan analyses (Right)ofβ-FG before and after 100 nM rapamycin application for 2 min. The line scans are as follows: Control, white line in images and black line in analysis graph; +Rapa, orange lines. (Scale bars: 10 μm.) (D) Rapamycin (Rapa)-induced

changes of cytosolic GFP fluorescence intensity in cells expressing LDR and translocatable β-FG subunits without CaV α1. Normalized cytosolic fluorescence intensities are shown as a function of time. F/F0, fluorescence divided by initial fluorescence (n = 5). (E) Confocal fluorescence images of cells expressing mitochondrial 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, compared with control. See SI Appendix, Fig. S2B for the ROIs selected for mitochondrial GFP intensity measurements in cells. (F) Confocal fluorescence images of cells expressing ER marker MR-Cb5 and β-FG without α1 before and after rapamycin addition. The selected ROIs are magnified on the Right where the arrow heads indicate 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 in control cells but significantly increased and colocalized with β2a-FG (arrow heads) in puncta after rapamycin addition. (Scale bars: 10 μm.) All images are representative of 7 to 10 cells in three independent experiments.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al. Downloaded by guest on September 27, 2021 of an FKBP–rapamycin–FRB complex (Fig. 1 A and B). We call cytoplasm and their overall subcellular distribution was not these FRB constructs anchors. In cells coexpressing β-FG iso- changed (SI Appendix, Fig. S5 A and B). With β3 subunits, the forms and the plasma membrane targeting anchor LDR, all of the cytosolic distribution actually increased at the lower ratio, per- cytosolic β-FG proteins translocated to the plasma membrane haps because fewer α1B subunits were successfully trafficked to with time constants of ∼25 to 30 s after addition of 100 nM the plasma membrane (SI Appendix, Fig. S5C). The expression of rapamycin (Fig. 1 C and D). Note that the β2a-FG isoform with α1B did not change the already high plasma membrane distri- two palmitoyl side chains in its N terminus was already well- bution of β2a and β2e isoforms. localized to the plasma membrane in control cells and showed The isotype-selective expression patterns of β subunits also no change during addition of rapamycin. Similarly, in cells contributed to a difference in electrophysiological properties of 2+ expressing the mitochondrial anchor Tom20-MR or the ER an- CaV2.2 channels. Throughout, we used Ba as the current carrier. chor MR-Cb5, all cytosolic β-FG isoforms rapidly translocated to SI Appendix,Fig.S4C compares representative current inactivation the mitochondria or the ER, respectively, with rapamycin addition traces of channels formed with different β-FG isoforms. With (Fig. 1 E and F and SI Appendix,Fig.S2). In cells expressing MR- plasma membrane-located β2a or β2e isoforms, the currents inac- Cb5, there was one difference: After rapamycin addition, the tivated very slowly whereas, with cytosolic β isoforms, they inacti- plasma membrane-localized palmitoylated β2a-FG congregated to vated rapidly (SI Appendix,Fig.S4D). In addition, we found that form puncta at ER-plasma membrane (ER-PM) junctions that the average peak CaV2.2 current density of cells expressing chan- became clearly visible in the confocal microscope within 2 min nels with plasma membrane-localized β isoforms was approximately (Fig. 1F and SI Appendix,Fig.S2D). Line scans of the plasma twofold higher than for those with cytosolic β isoforms (SI Ap- membrane revealed that β-FG and MR-Cb5 were colocalized in pendix,Fig.S4E). the puncta (Fig. 1G). Presumably, transient ER-PM junctions form often in the life of cells, and they are stabilized here when Interaction Between CaV α1B and β Subunits Is Stable in Cells. Is the rapamycin cross-links plasma membrane channels to an ER an- CaV α1–β interaction stable or dynamic? We approached this chor. Such control experiments validate translocatable β subunits question with our translocation technique. In cells coexpressing for use in intact living cells. the mitochondrial anchor Tom20-MR, we first tested the effects of rapamycin on CaV2.2 channels with β2 isoforms. As shown in Unlike Palmitoylated β2a-FG, the Electrostatically Tethered β2e-FG Fig. 2A, the palmitoylated β2a-FG, the palmitoylation-resistant Isoform Could Translocate to Mitochondria. Like the β2a subunit, mutant β2a(C3,4S), and nonpalmitoylated β2b all were mainly α β2e localizes to the plasma membrane even without CaV α1sub- localized at the plasma membrane in the presence of CaV 1B/ α δ units being present. It is not lipidated, but a polybasic region and a 2 1. Rapamycin treatment for 2 min did not trigger any re- NEUROSCIENCE hydrophobic tryptophan residue in the β2e N terminus make distribution of β2-FG isoforms from the plasma membrane to electrostatic and hydrophobic interactions with acidic phospho- mitochondria. Fig. 2C shows peak current-voltage relations for lipids in the plasma membrane (33–35) (SI Appendix,Fig.S3A). the different CaV2.2 channels in patch clamp electrophysiology. To probe this interaction, we used translocation in cells expressing The vertical axis reflects the maximum current amplitude, and β2e-FG and mitochondrial Tom20-MR. Without α1subunits,β2e- the position on the voltage axis reflects the voltage dependence FG was primarily localized at the plasma membrane (SI Appendix, of activation gating. With rapamycin addition, there were no Fig. S3B). When the cells were treated with rapamycin, the fluo- changes in the current amplitudes, in the activation curve (Fig. 2 rescence intensity of β2e-FG (green) decreased at the plasma C and E), or in the time course of inactivation gating (SI Ap- membrane and clearly increased in the mitochondrial region. By pendix, Fig. S6 A and B). Apparently, CaV α1B–β coupling is comparison, the fluorescence intensity of Tom20-MR (red) on the quite stable on a 2-min time scale in these intact cells. mitochondria did not change. Evidently, unlike for palmitoylated We also investigated α1B–β binding stability in cells expressing β2a-FG, rapamycin almost completely removes β2e-FG from the the ER anchor MR-Cb5. Consistent with Fig. 1 F and G, rapa- plasma membrane, translocating it to the mitochondria within mycin formed puncta of membrane-localized β2a-FG and MR- 2min.Whenβ2e-FG was coexpressed with ER-localized MR- Cb5 at ER-PM junctions (Fig. 2B). Results were similar for cells Cb5, the two proteins became colocalized in puncta at ER-PM expressing CaV2.2 with β2a(C3,4S)-FG or β2b-FG. Nevertheless, junctions after rapamycin addition (SI Appendix, Fig. S3C). These formation of puncta did not result in changes in the current data indicate that the electrostatic interaction of CaV β2e subunits amplitudes, in the activation curve, or in inactivation gating of i with the plasma membrane ( )isweakenoughtobeovercomeby CaV2.2 currents (Fig. 2 D and E and SI Appendix, Fig. S6 C and rapamycin-induced dimerization to distant and not very movable D). Apparently, β-FG and CaV α1B/α2δ1 translocate together mitochondria but (ii) is not overcome when rapamycin links it to laterally along the plasma membrane to the ER-PM junctions the more compliant ER that can readily make PM junctions sta- without change in the composition or gating properties of channel bilized by other molecules and that apparently does not pull hard complexes. The diagram in Fig. 2F illustrates movement of β enough to tear 2e subunits off the PM. CaV2.2 channels complexed with β-FG to the ER-PM junctions via rapamycin-induced heterodimerization. Results were similar α β The Phenotype of Interaction Between CaV 1B and Subunits in with P/Q-type CaV2.1 and R-type CaV2.3 channels, but not with β SI Cells. Next, we investigated the nature of CaV α1–β interactions T-type CaV3 channels, which do not complex with subunits ( in live cells using confocal microscopy. When expressed alone in Appendix, Fig. S7). tsA-201 cells, the β2a and β2e isoforms were mostly localized at Unlike the β2 isoforms, β1b-FG and β3-FG were found both in the plasma membrane whereas β1b, β2a(C3,4S), β2b, and β3 the cytoplasm and plasma membrane in cells expressing CaV α1B/ isoforms were distributed through the cytosol with some in the α2δ1(SI Appendix,Fig.S4B), consistent with previous reports (36– nucleus (SI Appendix,Fig.S4A). However, when they were 38). We examined whether some of those β subunits were interacting α A expressed together with CaV α1B/α2δ1 subunits, the β2a(C3,4S) with membrane 1B subunits. As shown in Fig. 3 ,rapamycin and β2b isoforms were found in the plasma membrane only or treatment rapidly translocated the cytosolic β-FG subunits to both in the plasma membrane and the cytoplasm in over 80% of mitochondria. After the translocation, significant residual green cells. This suggested that β2a(C3,4S) and β2b isoforms are as- fluorescence remained at the plasma membrane, implying that sociated with α1B/α2δ1 complexes in the plasma membrane some β1b-FG and β3-FG subunits were stably associated with α1B (SI Appendix, Fig. S4 A and B). However, in similar conditions, subunits. Interestingly, the currents were not affected by the ad- the β1b and β3 isoforms remained largely in the cytoplasm and dition of rapamycin (Fig. 3 B and C), suggesting that, once β1b or nucleus. Their cytoplasmic distribution was not due to a higher β3 subunits were bound to α1B, they remained as intact com- expression of β subunits compared with the α1B in the cells since, plexes, still modulating channel gating. In cells cotransfected with when the molar ratio of transfected β isoforms versus α1B was the ER anchor MR-Cb5, a significant number of puncta formed in decreased by 50%, most β1b subunits still were located in the ER-PM junctional regions after rapamycin addition (Fig. 3D).

Yeon et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 27, 2021 Fig. 2. Stable interaction of β2 subunits with α1B in intact live tsA-201 cells. (A and B) Representative confocal fluorescence images of cells expressing

CaV2.2 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). (Scale bars: 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. (C 2+ and 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 Ba currents are 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, in 10-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 in

MR-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). Data are mean ± SEM. (F) Diagram of rapamycin-induced puncta formation at ER-PM junctions. Based on our results, rapamycin triggers puncta formation by tethering 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.

Still, the current density and activation curve were not affected units at the plasma membrane already were fully complexed during rapamycin-induced translocation to puncta (Fig. 3 E–G), with β subunits. indicating again that some β1b and β3 subunits remain firmly as- sociated with α1B even after they are pulled into ER-PM junctions. A Dynamic Exchange of β Subunits Is Revealed When Two Different β We further confirmed with two-color confocal microscopy that Isoforms Are Expressed. In the experiments so far, we showed that, α1B pore-forming subunits migrate to puncta together with the when translocatable free β subunits are targeted to an organelle coupled β-FG. We used α1B labeled with YFP in the C terminus anchor by the addition of rapamycin, the existing α1B–β channel α β β ( 1B-YFP), 1b-FKBP-mCherry ( 1b-FmCh), and dark FRB- complexes do not give up their bound β subunits. This apparent Cb5. Rapamycin treatment induced formation of puncta con- stability contrasts with literature reports of β subunit exchange in taining both α1B-YFP and β1b-FmCh colocalized at the plasma β H I cells where one kind of subunit apparently can displace another. membrane (Fig. 3 and ). Similar behavior was found in cells To imitate the reported conditions, we compared rapamycin ef- coexpressing β3-FmCh rather than β1b-FmCh (SI Appendix, Fig. β β β fects in cells coexpressing CaV2.2 and translocatable 2e-FG alone S8). These observations suggest that some of the 1b and 3 with cells coexpressing Ca 2.2 and both β2e-FG and additional subunits interact stably with Ca α1B in intact cells, although V V nontranslocatable cytosolic β1b or β3 in a 1:1 ratio. With two kinds most of them are soluble in the cytosol, and that, with rapamycin, β the full channel complex migrates to ER-PM junctions along the of subunits, we would expect a mixed population of channels, α –β some containing the β2e-FG subunits and inactivating slowly and plasma membrane. The initial 1B interaction remained up to β β 2 h after rapamycin as evidenced by no changes in the current others containing 1b or 3 subunits and inactivating rapidly as is density and current inactivation (SI Appendix, Fig. S9). This sets seen in the black control traces in the middle and right columns of B an upper limit to the rate of dissociation and agrees well with in Fig. 4 . For comparison, the left column of the figure shows only β vitro work that says these complexes are very stable. slowly inactivating current when cells express only 2e-FG sub- Can recruiting cytosolic β subunits to the plasma membrane units. As indicated diagrammatically in Fig. 4A, if one kind of β enhance CaV2.2 currents? We triggered translocation of subunit could displace another, then when the pool of trans- cytosol-localized β-FG isoforms to the plasma membrane with locatable β2e-FG is sequestered to mitochondria by rapamycin, rapamycin (Fig. 3J). The translocation of β1b-FG and β3-FG the mixed β subunit population in the channel complexes might β isoforms was rapid (Fig. 3K). Nevertheless, the CaV2.2 currents gradually be replaced with 100% of the nontranslocatable 1b or did not change (Fig. 3 L and M), suggesting that all α1B sub- β3 subunits, and the characteristics of the recorded current would

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al. Downloaded by guest on September 27, 2021 NEUROSCIENCE

Fig. 3. Stable interaction of β1b and β3 isoforms with α1B in intact tsA-201 cells. (A) Representative confocal fluorescence images of cells expressing

CaV2.2 channels with Tom20-MR and β1b-FG (Top)orβ3-FG (Bottom) before and after a 2-min application of rapamycin (100 nM). Significant fluorescence signals are found at the PM after rapamycin addition as indicated by arrowheads. (B) Rapamycin effects on CaV2.2 channels in cells cotransfected with β1b-FG 2+ or β3-FG. Inward Ba currents are measured every 4 s by applying a depolarizing pulse to 10 mV. (C) Summary of rapamycin effects on CaV2.2 channel currents 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 confocal

images 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 magnified Below. 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. The

CaV2.2 currents are measured in whole-cell configuration, where cells are depolarized to +10 mV from −80 mV for 10 ms. (H) Confocal images of cells expressing 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 selected ROIs 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 strongly increased in puncta after rapamycin addition. (J) Schematic diagram of the rapamycin-induced translocation of cytosolic β-FG to the PM. Rapamycin triggers

formation 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 LDR before 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.)

eventually change to all fast inactivation. That indeed happened. through turnover, trafficking, or assembly and are diluting out In cells expressing CaV2.2 and β2e-FG alone, the slowly inacti- the existing population of slowly inactivating channels without vating current changed little after rapamycin addition for 20 min the supposed displacement of existing subunits? Since these ex- (Fig. 4 B and C). However, in cells expressing β2e-FG together planations both postulate a change in the number of functional with cytosolic β subunits, the proportion of CaV2.2 current that channels, we calculated the change of peak current for each cell inactivated rapidly increased significantly after addition of rapa- after 10 and 20 min of rapamycin (SI Appendix, Fig. S10C). The mycin (Fig. 4 B and C). This effect did not occur in the absence of summarized results show that, as is common for recordings from coexpression of the Tom20 mitochondrial anchor. Similar but CaV2.2 channels, the currents run down in time; however, the smaller effects were seen in experiments where rapamycin was average rate of decline does not depend on the expression of applied for only 10 min (SI Appendix,Fig.S10A and B). Such data either Tom20 or cytosolic β subunits (SI Appendix, Fig. S10D). would be consistent with the concept that cytosolic β1b or There is no evidence for incapacitation, turnover, or supplemen- β3 subunits are able to displace β2e subunits from existing channel tation of channels dependent on Tom20, rapamycin, or β subunit complexes in <20 min. subtype. We further tried to estimate the extent of change in the These observations merited more detailed examination. We proportion of rapidly and slowly inactivating channels in our ex- consider two alternative explanations: (i) could it be that slowly periments. We took the 500-ms time courses of recorded currents inactivating channels just become inoperative after rapamycin for pure β1b and β2e-FG subunits (without rapamycin) and then addition so thereby the relative fraction of rapidly inactivating mathematically combined these two templates to mimic the cur- channels appeared to rise in our normalized traces, or (ii) could rent time courses for mixtures exposed to rapamycin. This was it be that newly inactivating channels are being inserted rapidly done both for the cells in the 10-min experiment (SI Appendix,Fig.

Yeon et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 27, 2021 Acute Dissociation of β Subunits from CaV2.2 Channel Complexes Changes the Gating Properties. Next, we wanted to observe the functional effects of acute dissociation of β subunits from intact CaV channels. We weakened the interaction between the α-binding pocket (ABP) of β with the α-interaction domain (AID) of α1by mutation. Our double-mutant translocatable β subunits (hereafter named “mutant β-FG”) had methionine (M) and leucine (L) res- idues of the ABP replaced by alanine (A). These mutations reduce the AID-ABP interaction affinity by ∼250-fold (23), allowing spontaneous dissociation of mutant β subunits from CaV2.1 channel complexes within 5 min in inside-out patches (28). The following experiments showed that sequestration by rapamycin addition can dissociate these mutant β subunits from CaV2.2 channel complexes. The first experiment used only a membrane-localized I-II loop of α1B (including the AID domain) rather than the full α1B loop subunit. SI Appendix,Fig.S12shows the subcellular localization of mutant β-FG isoforms in cells coexpressing (i)theα1B I-II loop coupledtoLyn11 and CFP (called Lyn-I-II loop-CFP) with (ii) Tom20-MR. In control, the mutant isoforms of β1, β2a(C3,4S), and β2b were mainly distributed at the plasma membrane (presumably coupled to Lyn-I-II loop-CFP) with some in the cytosol whereas the mutant β2a forms were principally at the plasma membrane, pre- sumably through their palmitoylation. Interestingly, the mutant β3 forms were detected only in the cytosol. When the cells were β α Fig. 4. Dynamic interaction of 2e-FG with CaV 1B subunits in the presence treated with rapamycin, the mutant isoforms of β1b, β2a(C3,4S), of other cytosolic β isoforms. (A) Diagram of experiment to test if the in- β2b, and β3 translocated dramatically to the mitochondria from the teraction of α1B–β subunits is dynamic in the presence of other cytoplasmic β plasma membrane, suggesting that the mutant subunits are readily β isoforms. TsA-201 cells expressing CaV2.2 and rapamycin-translocatable 2e- dissociated from the AID domain and sequestered upon the ad- FG alone or together with other cytosolic β subunits were treated with 2+ dition of rapamycin. 20 nM rapamycin for 20 min, and the Ba currents before and after rapa- We turned to the functional effects of acute β subunit dissoci- mycin were measured. FRB is anchored to the mitochondrial (Mito) outer ation from intact CaV2.2 channels using the Tom20-MR anchor. membrane via mitochondrial import receptor subunit Tom20 (Tom20-MR). β (B) Representative Ba2+ current traces showing inactivation gating in cells In accordance with weakened mutant subunit binding, rapa- α α δ β β β mycin addition significantly decreased inward currents through expressing 1B/ 2 1 and 2e-FG alone or with 1b or 3 isoforms before β (Control) and 20 min after addition of rapamycin (20 nM). The CaV2.2 cur- CaV2.2 channels coupled with the mutated versions of 1b-FG, rents are measured in whole-cell configuration in cells depolarized to β2a(C3,4S)-FG, and β2b-FG subunits (Fig. 5 A and B). The effect +10 mV from −80 mV for 500 ms. (C) Summary of the rapamycin effects on took 10 to 30 s. Rapamycin had little effect on the current of β CaV2.2 current inactivation with β2e-FG alone or with other β isoforms. The channels with the plasma membrane-bound mutant 2a-FG sub- fraction of Ba2+ current remaining after 100-ms depolarization to +10 mV unit, and it had little effect on channels with mutant β3-FG, which (r100) is compared before (Control) and after rapamycin addition. Data are we suspect binds much more weakly to α subunits than the other mean ± SEM of three independent experiments (n = 5–7). *P < 0.05, **P < mutant β subunits. After rapamycin application, the inactivation 0.01, compared with control. 2+ kinetics of Ba currents in CaV2.2 channels with dissociable mutant β isoforms were almost as slow as for channels without any + β subunits (No β) (Fig. 5C). Thus, the fraction of Ba2 current A SI Appendix S11 ) and for those in the 20-min experiment ( ,Fig. remaining after 100-ms depolarizations to +10 mV (r100) was S11B). For the 10-min rapamycin experiment, the estimated increased with mutant isoforms of β1b-FG, β2a(C3,4S)-FG, and proportion of rapidly inactivating channels was ∼45% if the β2b-FG (Fig. 5D). Interestingly, the inactivation of channels with Tom20 anchor was not expressed and 68% if the Tom20 was mutant β3-FG was almost the same as the inactivation of channels coexpressed, and, for the 20-min experiment, the proportions without a β subunit. Upon rapamycin addition, we also found a were ∼36 to 84%, respectively. Thus, the half-time of the switch in significant decrease in maximum current amplitudes in channels kinetics might be roughly 15 min. We suggest that this is too fast with mutant versions of β1b-FG and β2b-FG (Fig. 5E). At the for normal turnover and represents instead the time course of a same time, the voltage dependence of activation gating of those displacement reaction with β2e-FG subunits being displaced by channels shifted to the right by up to ∼10 mV, approaching that of cytosolic β1b subunits on existing channels. channels without β subunits (SI Appendix,Fig.S13). Here, the One unanticipated observation in Fig. 4 B and C and SI Ap- dependence of tail-current amplitude on prepulse voltage serves pendix, Fig. S10 A and B requires mention. In the cells with two as a direct measure of the voltage dependence of activation gating. types of subunits but not expressing the Tom20 anchor, there was Before rapamycin addition, the tail current activation curve of β a surprising slowing in overall inactivation kinetics after contin- CaV2.2 channels in cells expressing the mutant 3-FG subunit was almost the same as for channels without any β subunits (No uous exposure to rapamycin for 10 and 20 min. This change can β be seen as a kinetic mismatch in fitting the rapamycin curves ), and adding rapamycin had no effect. Both observations sug- gest that the steady-state fraction of CaV2.2 channels bound to using templates that are from cells not treated with rapamycin in β SI Appendix mutant 3-FG subunits is quite small. Similarly, in confocal ex- , Fig. S11. We can only point out that, in every cell periments where cells expressing Lyn-I-II-CFP with mutant β1b- type, there are endogenous rapamycin targets, some with FKBP FG and MR-Cb5 formed puncta at the plasma membrane after and others with FRB domains, so addition of rapamycin still has rapamycin, the cells with mutant β3-FG did not (SI Appendix, effects on cells without overexpression of exogenous FRB do- Fig. S14). Nevertheless, we do see evidence of some α1B-mutant- mains. Thus, endogenous FKBP will become dimerized with the β3 interaction since the mutant β3 still induced a small increase FRB domain of the tyrosine kinase mammalian target of rapa- in the membrane expression of the α1B channel proteins com- mycin (mTOR) and the FKBP domain of β2e-FG subunits on pared with no-β cells (Fig. 5E). This increase was much less than channels will be dimerized with mTOR as well. The conse- for the other mutant subunits. We envision that, even if the steady- quences of these interactions would be beyond the scope of state binding fraction were, e.g., only 5 to 10%, the transiently this paper. bound complexes might be aided in trafficking to the surface before

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al. Downloaded by guest on September 27, 2021 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) Time

course of rapamycin effects on CaV2.2 peak currents in cells coexpressing diverse mutant β-FG isoforms + with or without Tom20-MR. Inward Ba2 currents are measured every 4 s by applying a depolarizing pulse to +10 mV. (B) Summary of the rapamycin effects on

CaV2.2 currents with mutant β-FG isoforms. Data are mean ± SEM of three independent experiments [n = 10 for β1b-FG; n = 8forβ2a-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 cells expressing α1B/α2δ1 without (No β) or with various mutant β-FG isoforms and Tom20-MR before (Con-

trol) and after rapamycin addition. The CaV2.2 cur- rents are measured in whole-cell configuration where cells are depolarized to +10 mV from −80 mV for 500 ms. (D) Summary of the rapamycin effects on

current inactivation of CaV2.2 channels with mutant + β-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) Peak NEUROSCIENCE + current-voltage (I-V) relations of Ba2 currents evoked by voltage steps to the indicated potential

(mV) in cells expressing CaV2.2 channels without (No β) or with mutant β-FG isoform and Tom20-MR. In- ward current density is measured with voltage steps from −50 to +40 mV, in 10-mV intervals, from a holding potential of −80 mV. Data are mean ± SEM.

the mutant β3 subunit dissociated again. The same effects of the β3 subunits were strongly inhibited by PI(4,5)P2 depletion, the various mutant β-FG subunits were also detected in experiments channels with mutant β3-FG showed only slight inhibition by PI with CaV2.3 channels. Thus, when the mutant β1b-FG was acutely (4,5)P2 depletion and no effects of rapamycin addition. This was recruited away from CaV2.3 channels, current amplitudes fell, and consistent with our suggestion that very few CaV α1B/α2δ1 the tail current activation curve was shifted to the right by ∼9mV channels are bound to mutant β3 subunits at any moment so they (SI Appendix,Fig.S15). Collectively, we find that, when the β behave like channels without β subunits (No β) (Fig. 6 C and D). subunit is acutely dissociated from CaV α1B, the current density Thus, bound β subunits are needed for PI(4,5)P2 regulation of decreases, the inactivation gating slows, and the activation curve CaV channels, and their subcellular location is critical for the shifts to more positive potentials. determination of PI(4,5)P2 sensitivity.

Dissociation of β Subunits from α1B Decreases the Phosphatidylinositol Discussion

4,5-Bisphosphate Sensitivity of CaV2.2 Channels. Plasma membrane Published work with recombinant expression systems shows that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] can be depleted CaV β subunits both increase cell surface expression of α1 subunits rapidly by the voltage-sensing phosphatase Dr-VSP, a poly- and modulate the biophysical properties of HVA CaV channel phosphoinositide phosphatase enzyme activated by depolarization gating (2–4). Here, we developed chemically translocatable (30, 31) of the membrane potential (39, 40). We used Dr-VSP to compare CaV β subunits to examine interactions between α1B and β subunits β the PI(4,5)P2 sensitivity of channels without and with various CaV- in CaV2.2 channel complexes and their real-time regulatory effects subunits. Without any β subunits, CaV α1B/α2δ1 channels were only in intact cells. We initially used the dimerization system to sequester weakly (∼10%) inhibited by PI(4,5)P2 depletion, similar to the re- all free subunits to the plasma membrane, ER, or mitochondria. sponses of CaV channels with the membrane-targeted β2a subunits Those results showed that the in-cell α1B–β interaction can look (Fig. 6 A and B). Nevertheless, inactivation gating of the β-less very stable for some of the β isotypes, especially those that are in- channels was relatively fast and more similar to that of channels trinsically membrane-localized. However, coexpressing additional with cytosolic β subunits (SI Appendix,Fig.S16). functionally different free β isoforms revealed that one β subunit can We measured the PI(4,5)P2 sensitivity of CaV2.2 channels with be exchanged for another, changing properties of the channel such β translocatable mutant isoforms before and after rapamycin as modulation by membrane depolarization and PI(4,5)P2 metabo- addition. As shown in Fig. 6C, currents in channels with unmu- lism. We review that evidence and at the end develop a hypothesis tated WT cytosolic β1b, β2b, or β3 subunits were significantly that reconciles these apparently contradictory observations. In ad- inhibited by PI(4,5)P2 depletion, and this current inhibition was dition, we showed that acute removal of β subunits reduces currents not affected by rapamycin addition. However, with loosely bound by up to 50% in less than 1 min and changes the gating kinetics. mutant cytoplasmic β1b or β2b subunits, rapamycin reduced the inhibition by PI(4,5)P2 depletion. Channels with membrane- Subcellular Distribution of β Subunits. Consider first the localiza- tethered WT or mutant β2a were insensitive to depletion of PI tion of β subunits in the absence of any α1 subunit. The β1b and (4,5)P2 (Fig. 6 C and D). Finally, whereas channels with WT β3 subunits are found in the cytosol, since they lack binding

Yeon et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 27, 2021 partners at the plasma membrane, whereas β2a and β2e subunits ciation was not always the same. It was greatest in channels with are tethered to the plasma membrane through two palmitoyl side mutant β1b subunits, where rapamycin decreased the currents by chains or nonselective electrostatic interactions, respectively ∼55%, and relatively less in channels with mutant β2 subunits. (33–35, 41). When α1 subunits are coexpressed with WT β sub- Interestingly, rapamycin did not change gating of channels units, all β subtypes showed partial or total membrane localiza- with mutant β3 subunits at all as if there is only infrequent tion, consistent with previous experiments in native cells and interaction between α1B and mutant β3 subunits. These results recombinant systems (36–38). We could demonstrate significant agree with earlier work showing that β subunits interact with association of the β1b and β3 subunits with α1B subunits at the α1 through both the high-affinity AID region and a lower af- plasma membrane since, when these membrane-localized β finity C terminus and that the interaction through the lower subunits were translocated and concentrated in punctate ER-PM affinity binding site is isotype-specific in the order β2 > junctions by rapamycin application, the α1B subunits went with β1b >>> β3 (13, 14). Similarly, we found that, when the in- them. Translocating the cytosolic free β1b or β3 subunits to the teraction between AID and ABP was weakened by double plasma membrane upon addition of rapamycin did not increase mutations, the rapamycin effect on current inhibition was CaV currents as if all of the α1B subunits at the plasma mem- greater in channels with mutant β1b than mutant β2. We also brane were already functionally partnered with a β subunit. suggest that the mutant β3 form interacts infrequently with Additional functional roles of the free cytosolic β1b and β3 iso- α1B subunits so most mutant β3 subunits are located in the forms are not clear, but it has been suggested that they could play cytosol and the current is small and not affected by rapamycin regulatory roles by interacting with other cytosolic proteins (42). addition. Rapamycin had little effect on the gating of Thus, a recent report showed that cytosolic β3 subunits reduce CaV2.2 channels with mutant β2a subunits, including current inositol 1,4,5-trisphosphate (IP3) formation and receptor-induced amplitude, voltage dependence of activation, and inactivation 2+ Ca release by physically forming β3/IP3 receptor complexes gating. Presumably, the palmitoyl side chains prevent the through the SH3-HOOK domain (43). dissociation of mutant β2a from CaV α1B on the addition of rapamycin. Real-Time Effects of β Subunit Dissociation on CaV2.2 Channel Gating. Our results established the real-time effects of β subunit dis- Previous studies of α1 and β subunit interactions in Xenopus sociation from CaV2.2 channels on gating properties in live cells. oocytes reported that selective mutations in the AID and ABP (i) We found that the current amplitude fell sharply, consistent interaction sites weaken the interaction and decrease delivery of with previous studies with mutant β subunits in inside-out patches, channel complexes to the plasma membrane (21, 23). The in- where the current decreased by 50% (28). (ii) We found that the teraction could be weakened by mutating two residues, Met and inactivation of CaV2.2 channels with mutant β subunits was slowed Leu, in the ABP site of the β subunit GK domain (28). In our and more similar to the inactivating current of CaV2.2 without β hands, such mutated β subunits have a lower affinity for the I-II subunits. At the same time, the voltage dependence of activation loop of α1B subunits and are easily dissociated from the channel gating was shifted to the right and became less steep. We found complex by rapamycin addition. The rapamycin-induced disso- similar 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 by membrane 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 are depolarized 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 (black

traces) and cells expressing Dr-VSP (red traces). (B) Summary of CaV2.2 inhibition by membrane depolarization in control and cells expressing Dr-VSP. Data are mean ± 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) Effects of 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-VSP activation. Peak tail currents are indicated by arrow heads. (D) Summary of Dr-VSP–induced current inhibition before and after rapamycin addition. The open

bar 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.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1809762115 Yeon et al. Downloaded by guest on September 27, 2021 Significance of β Subunits for CaV2.2 Channel Regulation by PI(4,5)P2. Lipscombe, Brown University, Providence, RI), CaV2.3 (from Terrance P. Plasma membrane PI(4,5)P2 is required for full activation of Snutch, University of British Columbia, Vancouver), Dr-VSP without EGFP (from B.H.), M -muscarinic receptor (from Guthrie Resource Center), the HVA CaV channels, and, when PI(4,5)P2 is depleted by stimu- 2 chemical translocation systems Lyn -FRB (LDR), Tom20-mCherry-FRB lation of Gq-coupled receptors or by externally expressed phos- 11 (Tom20-MR), and mCherry-FRB-Cb5 (MR-Cb5) (from Tobias Meyer, Stan- phatases, the CaV currents fall (44, 45). We previously found that ford University, Stanford, CA), and CaV2.2-YFP (from David T. Yue, Johns the PI(4,5)P2 sensitivity of HVA CaV channels is tightly con- β Hopkins University, Baltimore). Rat β1b subunit was cloned from rat brain trolled by the subcellular localization of CaV subunits (37, 46). β Thus, channels with plasma membrane-localized β subunits (β2a cDNA. To generate translocatable CaV -fluorescent protein fusions, FKBP was β ∼ amplified using PCR and subcloned into pEGFP-N1 or mCherry-N1 vectors or 2e) are only weakly ( 10%) inhibited by PI(4,5)P2 depletion β β (Clontech) using EcoRI and BamHI sites (SI Appendix,Fig.S1). To create -FKBP- whereas channels with cytosolic subunits are strongly (30 to fluorescent proteins, β isoforms were amplified using PCR and subcloned into 60%) inhibited (47). Here, we find that the intrinsic PI(4,5)P2 β β β pFKBP-EGFP-N1 vector using multicloning sites NheI and EcoRI for 1b, 2a, sensitivity of -less CaV2.2 channels is very weak and resembles β2a(C3,4S), and β2b and HindIII and EcoRI for β3. Point mutation of ABP in β that for channels with membrane-tethered subunits. These CaV β GK domain was achieved using the standard inverse PCR method. Primers results invite speculation that the subcellular positioning of for plasmids in this study are listed in SI Appendix,TableS1. flexible I-II domains of β-less α1B subunits is similar to that with membrane-targeted β subunits and that the soluble β subunits Cell Culture and Transfection. TsA-201 cells were cultured in 35-mm culture

augment PI(4,5)P2 regulation of CaV2.2 channels by pulling the dishes under standard growth conditions (37 °C and 5% CO2) in DMEM I-II loop of the α1B subunit in the cytosolic direction (48). (Sigma-Aldrich) supplemented with 10% FCS (HyClone; Thermo Scientific) and 0.2% penicillin/streptomycin (HyClone; Thermo Scientific) at 37 °C under Interaction Properties of α1B–β Subunits in Intact Cells and a Closing 5% CO2. For the expression of CaV channel subunits, cells were transiently cotransfected with various subunits using Lipofectamine 2000 (Invitrogen) Hypothesis. Our rapamycin-translocatable CaV β subunits are ideal according to the manufacturer’s protocol. Unless stated, the transfected for examining the stability of CaV α1–β interactions in intact cells. Using this system, we confirmed that, when a single type of β DNA mixture consisted of plasmids encoding α1, β, and α2δ1 at a 1:1:1 molar α –β ratio. For electrophysiological recordings and confocal microscopy experi- isoform is expressed, all WT 1B interactions are so strong that – they are not broken by sequestering most free β subunits from the ments, cells were plated onto poly-L-lysine coated coverslip chips 24 to 36 h after transfection and used within 12 to 24 h after plating. cytosol.InthepresenceofanERanchor,thePMcomplexesare clustered to ER-PM junctions without changes in channel elec- Electrophysiological Recordings. Whole-cell patch-clamp recordings were ac- + trophysiology, and the currents are not changed for up to 2 h after quired as described (47). Ba2 currents were recorded in whole-cell config-

rapamycin addition. Such results would imply that the binding of urations using a HEKA EPC-10 patch-clamp amplifier with PatchMaster NEUROSCIENCE CaV β subunits to α1B is almost irreversible, a conclusion con- β software (HEKA Elektronik). Some analysis used Fit Master software. Pipettes sistent with many previous papers showing that 2a does not were pulled from glass micropipette capillaries (World Precision Instruments) α dissociate from CaV 1A during inside-out patch clamp (28). In using a Flaming/Brown micropipette puller model P-97 (Sutter Instrument addition, CaV α1S/β1a and α1C/β1a do not dissociate in fluores- Co.) and had resistances of 1.5 to 3.5 megohms when filled with internal cence recovery after photobleaching (FRAP) experiments in solution. Series-resistance errors were compensated at >60%, and fast and skeletal muscle (26), and anti-β3 antibodies do not dissociate CaV slow capacitance was compensated for before the applied test pulse se- α1A/β3andα1B/β3 complexes in rat brain (49). However, the quences. Currents were sampled at 10 kHz and filtered at 3 kHz. For all results were different when we expressed two types of β subunit recordings, cells were held at −80 mV. All recordings were leak- and together, one membrane tethered and rapamycin translocatable capacitance-subtracted before analysis by use of a –P/5 protocol. The ex- ’ and the other primarily cytosolic and not rapamycin trans- ternal Ringer s solution contained 150 mM NaCl, 10 mM BaCl2, 1 mM MgCl2, locatable. Upon addition of rapamycin, the gating of channels 10 mM Hepes, and 8 mM glucose; pH was adjusted to 7.4 with NaOH and with mostly β2e subunits was changed to be like that of channels osmolarity of 321 to 350 mOsm. The internal pipette solution consisted of β 175 mM CsCl, 5 mM MgCl2, 5 mM Hepes, 0.1 mM 1,2-bis(2-aminophenoxy) with mostly cytosolic subunits, demonstrating a removal of ′ ′ translocatable β2e subunits from the channels in exchange for the ethane-N,N,N ,N -tetraacetic acid (BAPTA), 3 mM Na2ATP, and 0.1 mM β ∼ Na3GTP; the pH was adjusted to 7.4 with CsOH and osmolarity of 321 to other soluble subunits. We estimate an 15-min half-life for the 350 mOsm. All experiments were carried out at room temperature (20 to exchange. These results are consistent with previous observations β – 24 °C). The reagents were as follows: CsOH, BAPTA, Na2ATP, and Na3GTP of functional exchange of different subunits in living cells (24 (Sigma-Aldrich), and other chemicals (Merck). 27, 50, 51). Apparently, the α1B–β interaction is dynamic in a competitive binding situation. β Confocal Imaging. All imaging was performed with a Carl Zeiss LSM 700 As a hypothesis, we propose that dissociation of a WT confocal microscope. For live-cell imaging, a 40× (water) apochromatic ob- subunit from α1B is a concerted reaction requiring a second β jective lens at 1,024 × 1,024 pixels with digital zoom was used. For time subunit to displace it. It will not happen when there are no courses, 524 × 524 pixels were used. To analyze the time course of cytosolic competing subunits. Possibly, there are two parts to the in- fluorescence of various proteins, images were taken every 5 s in Zeiss ZEN im- teraction site: If a bound subunit transiently releases from one aging software. Membrane localization was assessed from the line intensity part, a competing subunit might capture the revealed partial site, histograms of plasma membrane in the images after acquisition. Line-scan tracks gaining a toehold to displace the bound subunit from the second were chosen to avoid the nucleus. Regions of interest (ROIs) were selected in the part. Presumably, it is not necessary for the competing subunit to cytosolic or organelle regions of cells, and quantitative analysis was performed be a different isoform, but, for our experiments, it is only when using the “profile“ and “measure“ tools in ZEN 2012 lite imaging software (Carl the displacement changes the gating properties of the channel Zeiss MicroImaging). All confocal images were transferred in TIFF format, and that our assay could document dissociation of one β subunit in raw data from the time course were processed with Microsoft Office Excel exchange for another. In most cells, more than two β isoforms 2012 and summarized in Igor Pro (WaveMetrics Inc.). are expressed at the same time, and thus they would compete with each other continuously. Relative expression levels of the β Statistical Analysis. Patch-clamp and imaging data were analyzed using Igor isoforms might determine the subunit composition in Ca Pro-6.37 (WaveMetrics), Excel 2016 (Microsoft), or ZEN 2012 (Carl Zeiss) V software programs. Statistics in text or figures represent mean ± SEM. Sta- channels and could contribute to dynamic modulation of the tistical comparisons were made by one-way or two-way ANOVA depending channels in a particular neuron. on 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 Materials and Methods ***P < 0.001. Plasmids, Cloning, and Mutagenesis. We used the following plasmids: rat β CaV1.2 and rat 2a (from William A. Catterall, University of Washington, ACKNOWLEDGMENTS. We thank Dr. William A. Catterall for reading and Seattle), rat β2a(C3,4S) and β2b (from Joyce Hurley, Indiana University, helpful discussion; and Lea M. Miller for technical assistance. We also thank Indianapolis), rat CaV2.2e[37b] (52) and rat α2δ and rat β3 (from Diane many laboratories for providing the plasmids. This work was supported by

Yeon et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 27, 2021 the following grants: a National Research Foundation of Korea (NRF) grant Program of the Ministry of Science, ICT and Future Planning (18-BD-06) (to funded by the Korea government [Ministry of Science, Information and B.-C.S.), the Korea Brain Research Institute (KBRI) basic research program Communications Technology (ICT), and Future Planning] (2016R1A2B4014253) funded by the Ministry of Science, ICT and Future Planning (18-BR-04-1) (to (to B.-C.S.), Basic Science Research Program Grant 2017R1A4A1015534 (to B.-C.S.), NIH Grant R37-NS08174 (to B.H.), and the Wayne E. Crill Endowed B.-C.S.), the Daegu Gyeongbuk Institute of Science and Technology R&D Professorship (to B.H.).

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