Stac adaptor regulate trafficking and function + of muscle and neuronal L-type Ca2 channels

Alexander Polster, Stefano Perni, Hicham Bichraoui, and Kurt G. Beam1

Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045

Contributed by Kurt G. Beam, December 5, 2014 (sent for review November 11, 2014; reviewed by Bruce P. Bean and Terry P. Snutch) + Excitation–contraction (EC) coupling in depends precisely regulated. As for other high-voltage activated Ca2 upon trafficking of CaV1.1, the principal subunit of the dihydropyr- channels, the auxiliary β-subunit is important for membrane traf- + idine (DHPR) (L-type Ca2 channel), to plasma membrane ficking (5, 6). However, it is clear that an additional factor(s) is regions at which the DHPRs interact with type 1 ryanodine recep- also crucial. Specifically, CaV1.1 expresses poorly (7) or not at all tors (RyR1) in the . A distinctive feature of in mammalian cells that are not of muscle origin (e.g., tsA201 2+ this trafficking is that CaV1.1 expresses poorly or not at all in cells), whereas the closely related cardiac/neuronal L-type Ca mammalian cells that are not of muscle origin (e.g., tsA201 cells), channel, CaV1.2, is readily expressed in such cells (8–10). The in which all of the other nine CaV isoforms have been successfully absence of RyR1 seems insufficient to account for the difficulty expressed. Here, we tested whether plasma membrane trafficking of expressing Ca 1.1 in tsA201 cells because Ca 1.1 expresses of Ca 1.1 in tsA201 cells is promoted by the adapter Stac3, V V V well in dyspedic myotubes, albeit producing less current per because recent work has shown that genetic deletion of Stac3 in channel (3). Here we explored the hypothesis that the adaptor skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and Ca 1.1 traffic together protein Stac3 is important for membrane trafficking of CaV1.1. V Stac3 is one of three isoforms of the Stac family of adaptor to the tsA201 plasma membrane, whereas CaV1.1 is retained in- + tracellularly when Stac3 is absent. Moreover, L-type Ca2 channel proteins, so named because they contain src homology 3 (SH3) and cysteine-rich domains. Based on quantitative PCR of adult function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantita- tively similar to that in myotubes, despite the absence of RyR1. mice, transcripts for Stac3 are found at high levels in skeletal

Although Stac3 is not required for surface expression of CaV1.2, muscle and at low levels in the cerebellum, forebrain, and eye, + the principle subunit of the cardiac/brain L-type Ca2 channel, whereas transcripts for Stac2 are found in these same, three,

Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate neuronally rich regions at levels comparable to those of Stac3 in of current inactivation, with Stac2 acting similarly. Overall, these skeletal muscle (11). Transcripts for Stac1 (often designated results indicate that Stac3 is an essential chaperone of CaV1.1 in simply as Stac) are present in cerebellum, fore-/midbrain, and skeletal muscle and that in the brain, Stac2 and Stac3 may signif- eye (at levels 1/10th or less those of Stac2) and also present in icantly modulate CaV1.2 function. the bladder and adrenal gland (11). In skeletal muscle, the ab- sence of Stac3 causes the failure of EC coupling in both mice 2+ Stac adaptor protein | L-type Ca channel | excitation–contraction coupling (11) and fish (12). Moreover, Stac3 appears to interact with CaV1.1 based on both localization at triad junctions (11, 12) and oltage-gated calcium channels serve to couple membrane coimmunoprecipitation (12). Our results now indicate that Stac3 Velectrical activity to various downstream signaling cascades, is a chaperone protein that binds directly to CaV1.1 and is nec- whose properties are strongly influenced by the spatial inter- essary for its plasma membrane expression. Stac3 and Stac2 also relationships between the calcium channels, effector proteins, bind to CaV1.2, which is not required for membrane trafficking and cellular organelles that comprise these signaling pathways. For example, in skeletal muscle the link between muscle exci- Significance tation and contraction [excitation–contraction (EC) coupling] depends upon triadic or dyadic junctions between the plasma + membrane and the sarcoplasmic reticulum (SR), with L-type Ca2 Voltage-gated calcium channels are essential for diverse cellu- lar functions. For example, Ca 1.1 channels trigger skeletal channels (dihydropyridine receptors, DHPRs) and type 1 ryanodine V and Ca 1.2 channels regulate neural receptors (RyR1) localized in junctional domains of the plasma V expression in response to neuronal activity. Thus, it is impor- membrane and the SR, respectively. The DHPRs, which contain tant to understand the cellular mechanisms that regulate de- Ca 1.1 as their principal subunit, are arranged into groups of four V livery of these channels to the plasma membrane and that (“tetrads”), evidently as a consequence of yet-to-be identified, govern calcium movements via the membrane-inserted chan- physical links between the DHPRs and the four homomeric nels. Here we show that the cellular adapter protein “Stac3” subunits of RyR1 (1, 2). These links are thought to be necessary participates in both processes. Specifically, Stac3 binds to both for the bidirectional functional interactions between the DHPR Ca 1.1 and Ca 1.2. This binding is essential for efficient de- and RyR1. In particular, the DHPR is thought to be con- V V livery of CaV1.1 to the plasma membrane, but not for CaV1.2. formationally coupled to RyR1 with the result that movements However, binding of Stac3, or the related protein Stac2, to of the CaV1.1 S4 helices in response to depolarization of the 2+ CaV1.2 causes a dramatic slowing of inactivation, thereby in- plasma membrane cause RyR1 to release Ca from the SR. In creasing calcium entry via Ca 1.2. addition to this orthograde signal, which is necessary for EC V

coupling, there is also a retrograde signal whereby the associa- Author contributions: A.P. and K.G.B. designed research; A.P., S.P., H.B., and K.G.B. per- 2+ tion with RyR1 causes the L-type Ca current via CaV1.1 to be formed research; A.P., S.P., H.B., and K.G.B. analyzed data; and A.P. and K.G.B. wrote + larger and to activate more rapidly. Thus, Ca2 currents are the paper. substantially reduced in size in dyspedic myotubes genetically Reviewers: B.P.B., Harvard Medical School; and T.P.S., University of British Columbia. null for RyR1 (3, 4). The authors declare no conflict of interest. Given the structural and functional interactions between 1To whom correspondence should be addressed. Email: [email protected]. CaV1.1-containing DHPRs and RyR1, it would seem necessary This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that the trafficking of CaV1.1 to the plasma membrane be 1073/pnas.1423113112/-/DCSupplemental.

602–606 | PNAS | January 13, 2015 | vol. 112 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1423113112 Downloaded by guest on September 27, 2021 but is functionally important, greatly slowing the inactivation of current. Results Fig. 1A compares the subcellular distribution of fluorescently tagged CaV1.2 and CaV1.1, after coexpression in tsA201 cells with the auxiliary β1a- and α2δ-subunits. CaV1.2 appeared to be associated with the surface membrane (Fig. 1A, Left), but CaV1.1 had a reticular distribution (Fig. 1A, Right) consistent with re- tention in the endoplasmic reticulum (ER) (13). Recently, Stac3 was reported to localize to plasma membrane/SR junctions (triads) in skeletal muscle, to be necessary for skeletal-type EC + coupling (not requiring entry of extracellular Ca2 ), and to be highly expressed in skeletal muscle but not in . Thus, we decided to test whether Stac3 would affect the traf- ficking of CaV1.1. Strikingly, the presence of Stac3 caused CaV1.1 to associate with the cell surface (Fig. 1B). To determine whether this surface-associated CaV1.1 was actually inserted into the plasma membrane, we used a monoclonal antibody to CaV1.1 (14). This antibody stained intact tsA201 cells coexpressing CaV1.1 and Stac3, but not cells expressing only CaV1.1 (Fig. S1). An obvious feature of cells like those illustrated in Fig. 1B was that Stac3, like CaV1.1, was associated with the surface. On the basis of photobleaching analysis, such surface binding was not an

intrinsic property of Stac3, but depended instead on CaV1.1 also Fig. 2. Stac3 promotes robust functional expression of CaV1.1 in tsA201 being present (Fig. S2). Moreover, even with neither β1a nor α2δ, cells. (A) Representative ionic currents and peak I–V relationships for YFP- = Stac3 appeared able to colocalize at the surface with CaV1.1 CaV1.1 in tsA201 cells either without Stac3 (open triangles, n 10) or with = – PHYSIOLOGY and to promote the insertion of CaV1.1 into the plasma mem- Stac3 (solid circles, n 14). (Right) Shown for comparison is the peak I V brane (Fig. S3). Taken together, these results strongly suggest relationship for dysgenic myotubes (null for endogenous CaV1.1), which were injected with cDNA encoding YFP-CaV1.1 (gray diamonds, n = 12). that Stac3 binds directly to CaV1.1 and that this binding is nec- essary for efficient delivery of Ca 1.1-containing DHPRs to the Calibrations: 5 pA/pF (vertical), 50 ms (horizontal). (B) Representative charge V movements (depolarizations from −50 mV to −30 mV, −10 mV, 10 mV, and plasma membrane. 30 mV) for YFP-CaV1.1 expressed in tsA201 cells either without or with Stac3. Whole-cell patch clamping was used to test whether CaV1.1 Calibrations: 1 pA/pF (vertical), 10 ms (horizontal). (Right) Average Q–V

was functional after its cotransfection with Stac3 in tsA201 cells. relationships for tsA201 cells expressing YFP-CaV1.1 with or without Stac3 As shown in Fig. 2, CaV1.1 in the absence of Stac3 produced and YFP-CaV1.1-expressing dysgenic myotubes are shown (symbols and + neither Ca2 currents nor membrane-bound charge movements numbers of cells as in A). The smooth curves (A and B, Right) are plots of Eqs. (gating currents), whereas, in the presence of Stac3, CaV1.1 1 and 2 (Materials and Methods) with the average parameters determined + produced robust Ca2 currents and charge movements. Fig. 2 also by fits of these equations to data from individual cells. Throughout, error ± compares the peak current–voltage and charge–voltage relation- bars represent SEM. ships for CaV1.1 in tsA201 cells with those of CaV1.1 expressed in dysgenic myotubes (“mdg,” null for endogenous Ca 1.1). We V two cell types because RyR1 is present in dysgenic myotubes but had expected that Ca 1.1 function would differ between the V not in tsA201 cells and because the absence of RyR1 in dyspedic myotubes (null for endogenous RyR1) has been shown to cause 2+ Ca currents via CaV1.1 to activate more rapidly (4) and to be severalfold smaller in relationship to charge movement (Gmax/ Qmax) (3, 4). However, double-exponential fits (Eq. 3) revealed no significant differences between tsA201 cells and dysgenic + myotubes in the rate of Ca2 current activation (Fig. 3). Addi- tionally, based on fits of Boltzmann expressions (Eqs. 1 and 2), there were no significant differences between the two cell types in the magnitude of the ratio Gmax/Qmax (Fig. 3). Thus, the function of CaV1.1 coexpressed with Stac3/β1a/α2δ in tsA201 cells is remarkably similar to that of CaV1.1 as part of the DHPR complex in myotubes. In addition to binding to the DHPR, it has been suggested that Stac3 may also bind to RyR1 and thus serve as a functional link between the two proteins (11, 12). As a test of this possibility, we determined whether Stac3-YFP had the distribution expected for targeting to plasma membrane/SR junctions, which are present with a punctate distribution in both dyspedic (15, 16) and dys- genic myotubes (17). Stac3-YFP had a punctate distribution in Fig. 1. CaV1.1 is retained in the ER of tsA201 cells but traffics to the surface dyspedic myotubes (Fig. 4A, Left), which contain DHPRs but when Stac3 is present. (A) Subcellular distribution of YFP-labeled CaV1.2 and lack RyR1. This result is consistent with the apparent interaction CaV1.1 in tsA201 cells. (Scale bars, 5 μm.) (B) Fluorescence images demon- strating that CFP-Ca 1.1 (green) and Stac3-YFP (red) traffic to the surface between Stac3 and DHPRs in tsA201 cells (Fig. 1B, extended V data in Fig.2). By contrast, Stac3-YFP had a diffuse distribution after coexpression in tsA201 cells. (Scale bar, 2 μm.) The auxiliary β1a- and α2δ-subunits were also present here and in all other tsA201 cell experiments, in dysgenic myotubes (Fig. 4A, Right), which contain RyR1 but unless otherwise mentioned. not CaV1.1. This latter result could mean either that Stac3-YFP

Polster et al. PNAS | January 13, 2015 | vol. 112 | no. 2 | 603 Downloaded by guest on September 27, 2021 Discussion If, as our results suggest, Stac3 is required for efficient mem- brane trafficking of CaV1.1, then it stands to reason that EC coupling would fail in animals that lack it. However, it seems likely that its continued presence is required for maintaining the EC coupling apparatus in a functional state. In particular, EC coupling is greatly impaired in muscle fibers of Stac3-null zebrafish embryos at 48 h postfertilization (hpf) even though there is a near-normal level of triadic CaV1.1 (12). Moreover, these Stac3-null embryos displayed some motile activity at 2 hpf, which could be attributed to the presence of Stac3 translated from maternally deposited mRNA. Thus, one could postulate that the maternally derived Stac3 supported the triadic insertion of CaV1.1, but that the subsequent loss of Stac3 protein caused CaV1.1 to become inoperative for EC coupling. An important goal for future research will be to identify the domains of CaV1.1 and Stac3 that are important for their interactions with one 2+ Fig. 3. L-type Ca currents are similar for YFP-CaV1.1 expressed in dysgenic another. It will also be important to determine whether the 2+ =+ + myotubes and tsA201 cells. (A) Comparison of peak Ca currents (Vtest 40 modulation of Ca2 channel function that we have shown to be mV) in a naive dyspedic (RyR1-null) myotube; a dysgenic myotube trans- produced by Stac2 and Stac3 also occurs for Stac1 and to de- fected with YFP-Ca 1.1; and a tsA201 cell cotransfected with YFP-Ca 1.1, α δ, V V 2 termine whether such modulation actually occurs in specific β1a, and Stac3. Calibrations: 5 pA/pF, vertical; 50 ms, horizontal. (B and C) + Activation was fitted with a double-exponential function (Eq. 3) for Ca2 neuronal populations. currents at +40 mV in naive dyspedic myotubes (open bars, n = 10), YFP- The ability to achieve high-level expression of CaV1.1 in CaV1.1 expressing dysgenic myotubes (solid bars, n = 10), and in YFP-CaV1.1/ tsA201 cells provides a new experimental tool that will facilitate Stac3 expressing tsA201 cells (shaded bars, n = 12). Based on one-way the study of skeletal muscle DHPRs without the time and ex- ANOVA, there was no statistically significant difference between the tsA201 pense involved in maintaining colonies of animals heterozygous cells and dysgenic myotubes in either the time constants (B, τslow, P = 0.461; τ = = = for mutations of DHPR subunits. The relative absence of con- fast, P 0.421) or amplitudes (C, Aslow, P 0.394; Afast, P 0.400) of the slow taminating currents is also an advantage. For example, the ability and fast components of current activation. (D) Peak current–voltage (peak I–V) and charge–voltage (Q–V) relationships were fitted with Eqs. 1 and 2, to study how mutations alter the voltage dependence of CaV1.1 charge movements is hampered in dysgenic myotubes by the respectively, to yield values of Gmax, Qmax, and Gmax/Qmax for individual cells. Mean values (±SEM) are shown for dyspedic myotubes (open bars, n = 6), variable presence of gating currents arising from other ion

YFP-CaV1.1 expressing dysgenic myotubes (solid bars, n = 6), and YFP-CaV1.1/ channels, whereas background gating currents are essentially Stac3 expressing tsA201 cells (shaded bars, n = 8). Data shown were obtained nonexistent in tsA201 cells (Fig. 2B, Right). Moreover, experi- only from cells in which both peak I–V and Q–V relationships were mea- ments on tsA201 cells may provide unexpected insights. For sured. The Gmax/Qmax ratios for dyspedic and dysgenic myotubes were not example, it was previously hypothesized that the loss of a “cur- corrected for any background charge (unrelated to CaV1.1) because this was rent-enhancing” signal from RyR1 to CaV1.1 accounted for the small in the current experiments: 0.87 ± 0.09 nC/μF, n = 7, measured at 2+ +40 mV in naive dysgenic myotubes. reduced size of Ca currents in RyR1-null myotubes (3). However, this hypothesis appears to be incompatible with the 2+ near equivalence of CaV1.1 Ca currents in tsA201 cells and 2+ does not bind to RyR1 or that the RyR1 binding sites are oc- dysgenic myotubes (Figs. 2 and 3). Thus, it may be that Ca cluded in dysgenic myotubes by endogenous Stac3. Thus, we also currents via CaV1.1 are inhibited by other junctional proteins tested for an interaction between Stac3-YFP and RyR1 after unless RyR1 is present to relieve this inhibition. In principle, this expression of both in tsA201 cells. Fig. 4B illustrates a cell in idea and other aspects of DHPR signaling in skeletal muscle which the two proteins were expressed at high levels and shows images obtained before and after bleaching YFP within the in- dicated region of interest. This bleaching would not have been expected to affect any Stac3-YFP that remained bound to RyR1. Thus, the absence in the postbleach image of YFP fluorescence in regions containing RyR1 argues against a stable interaction between Stac3 and RyR1. This conclusion differs from that drawn from previous work on muscle tissue, in which Stac3 was found to coimmunoprecipitate with RyR1 (12). It is possible that we failed to detect such an interaction because the C-terminal YFP tag in- terfered with the binding of Stac3 to RyR1. Alternatively, it may be that the previously described coimmunoprecipitation was a conse- quence of a ternary complex of Stac3, the DHPR, and RyR1. As described above, the membrane trafficking of CaV1.2 in tsA201 cells does not depend on the presence of Stac proteins (Fig. 1A). However, low levels of Stac3 transcripts and high levels of Stac2 are present in the cerebellum and fore-/midbrain (11), Fig. 4. Stac3 does not appear to bind to RyR1 in either myotubes or tsA201 where CaV1.2 is also present (18). Thus, we tested whether Stac3 cells. (A) Stac3-YFP (red) had a punctate distribution in dyspedic (RyR1 null) and/or Stac2 might interact with CaV1.2. As shown in Fig. 5A, both Stac3-YFP and Stac2-YFP colocalized with CFP-Ca 1.2 myotubes (Left) and a diffuse distribution in dysgenic (CaV1.1 null) myotubes V (Right), indicative of a lack of binding to RyR1 because punctate plasma at the surface of tsA201 cells. Furthermore, this interaction alters membrane/SR junctions are present in both types of myotubes. (B) Images of CaV1.2 function. Specifically, in addition to causing modest changes a tsA201 cell coexpressing CFP-RyR1 (green) and Stac3-YFP (red) before (Left) 2+ in Ca current magnitude, both Stac2 and Stac3 caused a profound and after (Right) bleaching of YFP within the indicated polygon. Note that slowing of the rate of inactivation (Fig. 5B). CFP-RyR1 is localized in stacks of elongated ER. (Scale bars, 5 μm.)

604 | www.pnas.org/cgi/doi/10.1073/pnas.1423113112 Polster et al. Downloaded by guest on September 27, 2021 fragment of pEYFP-C1 that had been digested with the same enzymes.

Digestion of the plasmid β1a-YFP (24) Acc65I/BsrGI, followed by self-ligation of the compatible ends, was used to produce the expression vector for un-

labeled β1a. Unlabeled α2δ-subunit was kindly provided by William A. Sather (University of Colorado).

Primary Skeletal Culture and cDNA Microinjection. All procedures involving mice were approved by the University of Colorado Denver– Anschutz Medical Campus Institutional Animal Care and Use Committee. Myoblasts were prepared from newborn dysgenic mice, homozygous for

absence of CaV1.1 (25), or newborn dyspedic mice, homozygous for absence of RyR1 (26) as described before (27). For electrophysiological experiments, myoblasts were plated into 35-mm, plastic culture dishes (Falcon) coated with ECL (Millipore). Myoblasts destined for imaging were plated into 35-mm cul- ture dishes with ECL-coated glass-coverslip bottoms (MatTek). Cultures were

grown for 4–5 d in a humidified 37 °C incubator with 5% (vol/vol) CO2 in high- glucose Dulbecco’s modified Eagle medium (DMEM) (Mediatech), supple- mented with 10% (vol/vol) FBS and 10% (vol/vol) horse serum (both from HyClone Laboratories). After 4–5 d, this medium was replaced with differen- tiation medium [DMEM supplemented with 2% (vol/vol) horse serum]. Two to 4 d following the shift to differentiation medium, single nuclei were microinjected with plasmid cDNA (200 ng/μL in water). Forty-eight hours after injection, expressing cells were identified on the basis of YFP fluorescence.

tsA201 Cell Culture and Expression of cDNA. tsA201 cells were propagated in Fig. 5. Stac3 and Stac2 associate with Ca 1.2 in tsA201 cells and slow its V high-glucose DMEM supplemented with 10% (vol/vol) FBS and 2 mM glu- inactivation rate. (A) Images of tsA201 cells coexpressing CFP-CaV1.2 (green) μ tamine in a humidified incubator with 5% (vol/vol) CO2. Cells were plated at and either Stac3-YFP (red, Top) or Stac2-YFP (red, Bottom). (Scale bars, 5 m.) 5 + a density of 2 × 10 cells in 35-mm dishes and transfected by Lipofectamine (B, Left) Whole-cell Ca2 currents in tsA201 cells expressing YFP-Ca 1.2 V 2000 (Life Technologies) 24 h later with equal concentrations (1 μg/μL) of without Stac 2 or Stac3 (Vtest =+20 mV) and YFP-CaV1.2 with either Stac3 various combinations of YFP-CaV1.2, CFP-CaV1.2, YFP-CaV1.1, CFP-CaV1.1, un- (V =+30 mV; vertically scaled 1.7-fold) or Stac2 (V =+20 mV; vertically PHYSIOLOGY test test labeled Ca 1.1, CFP-RyR1, β , α δ, unlabeled Stac3, Stac3-DsRed, Stac3-YFP, scaled 0.75-fold). Vertical calibration, 10 pA/pF (nonscaled current); hori- V 1a 2 and unlabeled Stac2 cDNA. Six hours following transfection, cells were removed zontal, 50 ms. Peak I–V relationships (Center) and the fraction-of-peak cur- from the dish, using Trypsin EDTA (Mediatech), and replated at ∼1 × 104 cells rent at 150 ms (Right) are indicated for YFP-Ca 1.2 without Stac2 or Stac3 V per 35-mm dish to obtain isolated cells for electrophysiological recording. (gray symbols, +20 mV, n = 11) and YFP-Ca 1.2 with either Stac3 (black V Seventy-two hours following transfection, positively transfected cells were symbols, +30 mV, n = 9) or Stac2 (white symbols, +20 mV, n = 10). identified by fluorescence and used for electrophysiology or imaging.

could be addressed by using tsA201 cells to manipulate the set Immunostaining. Transfected tsA201 cells were washed twice with PBS con- taining 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4,pHto of junctional proteins present in addition to CaV1.1 and Stac3. 7.4 with HCl, and fixed in paraformaldehyde [4% (vol/vol) in PBS] for 20 min Of course, there are many questions about signaling inter- at room temperature. After fixation, the cells were washed three times with actions between the DHPR and RyR1 that will continue to PBS and incubated in 10% (vol/vol) goat serum/1% BSA/PBS for 60 min at

require the use of skeletal muscle. However, even those ex- room temperature. Incubation in mouse CaV1.1 subunit primary antibody periments are likely to benefit from insights obtained in IIF7 (1:500 diluted in 1% BSA/PBS, kindly provided by Kevin P. Campbell, experiments on tsA201 cells. University of Iowa, Iowa City, IA) was overnight at 4 °C, after which the cells were washed three times for 10 min with 1% BSA/PBS. Cells were then in- Materials and Methods cubated in Alexa Fluor 568-labeled secondary antibody (diluted 1:300 in 1% BSA/PBS) for 1h at room temperature, washed with 1% BSA/PBS (3 × 10 Molecular Biology. The construction of the expression plasmids for GFP-tag- min, with gentle agitation), and imaged. ged, CFP-tagged, YFP-tagged, or unlabeled CaV1.1 was described previously (19, 20). The coding sequence of Ca 1.2 (21) was inserted “in frame” and V Confocal Microscopy and Photobleaching. Myotubes or tsA201 cells were downstream of enhanced yellow or cyan fluorescent protein in a mamma- superfused with Rodent Ringer’s solution (146 mM NaCl, 5 mM KCl, 2 mM lian expression vector (pEYFP-C1, pECFP-C1; Clontech) to create YFP-Ca 1.2 V CaCl , 1 mM MgCl , 10 mM Hepes, pH 7.4, with NaOH) and examined using and CFP-Ca 1.2. The plasmid CFP-RyR1, labeled at its N terminus with en- 2 2 V a Zeiss LSM 710 confocal microscope. Images were obtained as a single op- hanced cyan fluorescent protein, was derived from the original GFP-RyR1 tical slice with a 40× [1.3 numerical aperture (NA)] or 63× (1.4 NA) oil im- plasmid (22, 23) by excising the RyR1 encoding sequence from GFP-RyR1 and mersion objective. Excitation and emission, respectively, were 440 nm (diode inserting it into the multiple-cloning site of ECFP-C1 (Clontech). PCR with laser) and 465–495 nm for CFP, 488 nm (argon laser) and 498–544 nm for ′ ′ the primers [forward (fw)] 5 -GAATTCATGACAGAAAAGGAAGTGGTG-3 and GFP, 514 nm (argon laser) and 530–565 nm for YFP, 543 nm (HeNe laser), and ′ ′ [reverse (rev)] 5 -GGATCCCAAATCTCCTCCAGGAAGTCG-3 was used to in- either 565–721 nm for DsRed or 596–664 nm for Alexa Fluor 568. Relative to ′ ′ troduce EcoRI (5 ) and BamHI (3 ) sites flanking the coding sequence for full power output, the excitation was attenuated to ∼4% (440 nm), ∼2% Stac3 (gene ID 237611; plasmid kindly provided by Eric N. Olson, University (488 nm and 514 nm), ∼15% (543 nm, DsRed), and ∼3% (543 nm, Alexa Fluor of Texas Southwestern Medical Center, Dallas), from which the EcoRI-BamHI 568). For photobleaching of Stac3-YFP in tsA201 cells, a prebleach image was fragment was inserted into EYFP-N1 (Clontech) to create Stac3-YFP. To obtained as described above. Within this image, a smaller region of interest create Stac3-DsRed, the Stac3 fragment from Stac3-YFP was inserted into was designated to avoid the cell surface and repeatedly scanned for 15–45 s pDsRed1-N1 (Clontech), using the restriction enzymes HindIII and BamHI. To with nonattenuated 514-nm excitation. About 5–10 s after completion of provide an unlabeled Stac3 construct with a comparable expression level, the bleaching, a postbleach image was then obtained with the same settings Stac3-YFP was digested with NdeI and BamHI, and the fragment containing as those of the prebleach image. the Stac3 sequence was then ligated to the 3,575-bp fragment of pEYFP-C1 + that had been digested with the same enzymes. PCR with the primers (fw) Measurement of L-Type Ca2 Currents and Intramembrane Charge Movements. 5′-CTGAGGTACCAACCATGACCGAAATGAGCGAGAAG-3′ and (rev) 5′-GTGGT- All experiments were performed at room temperature (∼25 °C). Pipettes ACCTAGATCTCTGCCAAGGAGTCG-3′ was used to introduce KpnI sites and a (∼2.0 MΩ) were fabricated from borosilicate glass and were filled with in-

stop codon flanking the coding sequence for Stac2 (gene ID 217154; OriGene ternal solution, which consisted of 140 mM Cs-aspartate, 10 mM Cs2-EGTA, Technologies), from which the KpnI-KpnI fragment was inserted into EYFP-N1. 5 mM MgCl2, and 10 mM Hepes, pH 7.4, with CsOH. The bath solution Afterward this construct was digested with NdeI and BamHI, and the frag- contained 145 mM tetraethylammonium-Cl, 10 mM CaCl2,and10mM ment containing the Stac2 sequence was then ligated to the 3,575-bp Hepes, pH 7.4, with NaOH. For electrophysiological experiments on

Polster et al. PNAS | January 13, 2015 | vol. 112 | no. 2 | 605 Downloaded by guest on September 27, 2021 myotubes, 0.003 mM tetrodotoxin (TTX) and 0.1 mM N-benzyl-p-toluene = Qmax sulphonamide (BTS) were added to the bath solution. To isolate L-type Qon , [2] f1 + exp½ðVQ − VÞ=kQg currents in myotubes, voltage was stepped from the holding potential (−80 − mV) to 20 mV for 1 s to inactivate endogenous T-type current. For mea- where Qmax is the maximal Qon, VQ is the potential causing movement of surement of intramembrane charge movements attributable to CaV1.1, 0.1 half the maximal charge, and kQ is a slope parameter. The activation phase M LaCl3 and 0.5 M CdCl2 were added to the bath solution and a prepulse of macroscopic ionic currents was fitted as described in ref. 4, using the 2+ protocol (28) was used to inactivate T-type Ca channels and sodium exponential function channels. Electronic compensation was used to reduce the effective series     < Ω < μ − − resistance to 5M (time constant 400 s). Linear components of leak and = t + t + − IðtÞ Afast exp Aslow exp C, [3] capacitive current were corrected with P/4 online subtraction protocols. τfast τslow Filtering was at 2–5 kHz and digitization was either at 10 kHz (L-type cur- rents) or at 25 kHz (charge movements). Cell capacitance was determined by where I(t) is the current at time t after the depolarization, Afast and Aslow are integration of a transient elicited by stepping from the holding potential the steady-state current amplitudes of each component with their respective τ τ (−80 mV) to −70 mV, using Clampex 8.2 (Molecular Devices), and was used to time constants of activation ( fast and slow), and C represents the peak normalize charge movements (nC/μF) and ionic currents (pA/pF). Peak cur- current. Fractional inactivation of current in 150 ms (R150) was determined by rent–voltage (I–V) curves were fitted according to dividing the peak current into the current 150 ms after the peak.

G ðV − V Þ I = È max À rev Á ÃÉ, [1] Analysis. Figures were made using the software program SigmaPlot (version 1 + exp − V − V1=2 kG 11.0; SSPS). All data are presented as mean ± SEM. Statistical comparisons were made by one-way ANOVA. where I is the peal current for the test potential V, Vrev is the reversal po- 2+ tential, Gmax is the maximum Ca channel conductance, V1/2 is the half- ACKNOWLEDGMENTS. We thank Ms. O. Moua for expert technical assis- maximal activation potential, and kG is the slope factor. Plots of the integral tance. This work was supported by grants from the National Institutes of of the ON transient (Qon) of intramembrane charge movement as a function Health (AR055104) and the Muscular Dystrophy Association (MDA277475) of test potential (V) were fitted according to (to K.G.B.).

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