Stac adaptor proteins 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 skeletal muscle 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 receptor (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 sarcoplasmic reticulum. 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 protein 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 muscle contraction and Ca 1.2 channels regulate neural gene 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 cardiac muscle. 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.