Alexander Polster

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

Significance Voltage-gated calcium channels are essential for diverse cellular functions. For example, CaV1.1 channels trigger skeletal muscle contraction and CaV1.2 channels regulate neural gene expression in response to neuronal activity. Thus, it is important to understand the cellular mechanisms that regulate delivery of these channels to the plasma membrane and that govern calcium movements via the membrane-inserted channels. Here we show that the cellular adapter protein “Stac3” participates in both processes. Specifically, Stac3 binds to both CaV1.1 and CaV1.2. This binding is essential for efficient delivery of CaV1.1 to the plasma membrane, but not for CaV1.2. However, binding of Stac3, or the related protein Stac2, to CaV1.2 causes a dramatic slowing of inactivation, thereby increasing calcium entry via CaV1.2. Excitation–contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca2+ channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that CaV1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine CaV isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of CaV1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and CaV1.1 traffic together to the tsA201 plasma membrane, whereas CaV1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca2+ channel function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of CaV1.2, the principle subunit of the cardiac/brain L-type Ca2+ channel, Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of CaV1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate CaV1.2 function.

Stac3 has a direct role in skeletal muscle-type excitation-contraction coupling that is disrupted by a myopathy-causing mutation.

Significance Recent work showed that absence of the protein Stac3 (SH3 and cysteine-rich domain 3) caused a failure of excitation–contraction (EC) coupling in skeletal muscle but not whether this failure was because the trafficking of other key proteins was altered or because Stac3 plays a direct role in coupling CaV1.1 (the “sensor” of excitation) to RyR1 (type 1 ryanodine receptor, the Ca2+ release channel). Here we show that reduced expression of CaV1.1 could not account for the loss of EC coupling. Ca2+ release was fully restored by WT Stac3 but only marginally by Stac3 bearing a point mutation causing Native American myopathy. Thus, Stac3 seems to be involved directly in the coupling of CaV1.1 to RyR1. In skeletal muscle, conformational coupling between CaV1.1 in the plasma membrane and type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) is thought to underlie both excitation–contraction (EC) coupling Ca2+ release from the SR and retrograde coupling by which RyR1 increases the magnitude of the Ca2+ current via CaV1.1. Recent work has shown that EC coupling fails in muscle from mice and fish null for the protein Stac3 (SH3 and cysteine-rich domain 3) but did not establish the functional role of Stac3 in the CaV1.1–RyR1 interaction. We investigated this using both tsA201 cells and Stac3 KO myotubes. While confirming in tsA201 cells that Stac3 could support surface expression of CaV1.1 (coexpressed with its auxiliary β1a and α2-δ1 subunits) and the generation of large Ca2+ currents, we found that without Stac3 the auxiliary γ1 subunit also supported membrane expression of CaV1.1/β1a/α2-δ1, but that this combination generated only tiny Ca2+ currents. In Stac3 KO myotubes, there was reduced, but still substantial CaV1.1 in the plasma membrane. However, the CaV1.1 remaining in Stac3 KO myotubes did not generate appreciable Ca2+ currents or EC coupling Ca2+ release. Expression of WT Stac3 in Stac3 KO myotubes fully restored Ca2+ currents and EC coupling Ca2+ release, whereas expression of Stac3W280S (containing the Native American myopathy mutation) partially restored Ca2+ currents but only marginally restored EC coupling. We conclude that membrane trafficking of CaV1.1 is facilitated by, but does not require, Stac3, and that Stac3 is directly involved in conformational coupling between CaV1.1 and RyR1.

Sequence differences in the IQ motifs of Cav1.1 and Cav1.2 strongly impact calmodulin binding and calcium-dependent inactivation

The proximal C terminus of the cardiac L-type calcium channel (CaV1.2) contains structural elements important for the binding of calmodulin (CaM) and calcium-dependent inactivation, and exhibits extensive sequence conservation with the corresponding region of the skeletal L-type channel (CaV1.1). However, there are several CaV1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding CaV1.2 residues, including three of the “IQ motif.” To investigate the functional significance of these residue differences, we used native gel electrophoresis and expression in intact myotubes to compare the binding of CaM to extended regions (up to 300 residues) of the C termini of CaV1.1 and CaV1.2. We found that in the presence of Ca2+ (either millimolar or that in resting myotubes), CaM bound strongly to C termini of CaV1.2 but not of CaV1.1. Furthermore, replacement of two residues (Tyr1657 and Lys1662) within the IQ motif of a C-terminal CaV1.2 construct with the divergent residues of CaV1.1 (His1532 and Met1537) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in CaV1.1 caused a gain of CaM binding. In full-length CaV1.2, substitution of these same two divergent residues with those of CaV1.1 (Y1657H, K1662M) eliminated calcium-dependent inactivation of the heterologously expressed channel. Thus, our results reveal that a conserved difference between the IQ motifs of CaV1.2 and CaV1.1 has a profound effect on both CaM binding and calcium-dependent inactivation.

Fluorescence Resonance Energy Transfer (FRET) Indicates that Association with the Type I Ryanodine Receptor (RyR1) Causes Reorientation of Multiple Cytoplasmic Domains of the Dihydropyridine Receptor (DHPR) α1S Subunit

Background: In skeletal muscle, DHPR cytoplasmic domains are thought to couple membrane depolarization to Ca2+ release via RyR1. Results: The presence of RyR1 alters FRET between donor/acceptor pairs in cytoplasmic domains of the DHPR α1S subunit. Conclusion: Interaction with RyR1 causes rearrangement of α1S cytoplasmic domains. Significance: Multiple cytoplasmic domains of α1S may be involved in the interaction with RyR1. The skeletal muscle dihydropyridine receptor (DHPR) in the t-tubular membrane serves as the Ca2+ channel and voltage sensor for excitation-contraction (EC) coupling, triggering Ca2+ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). The two proteins appear to be physically linked, and both the α1S and β1a subunits of the DHPR are essential for EC coupling. Within α1S, cytoplasmic domains of importance include the I-II loop (to which β1a binds), the II-III and III-IV loops, and the C terminus. However, the spatial relationship of these domains to one another has not been established. Here, we have taken the approach of measuring FRET between fluorescent proteins inserted into pairs of α1S cytoplasmic domains. Expression of these constructs in dyspedic (RyR1 null) and dysgenic (α1S null) myotubes was used to test for function and targeting to plasma membrane/SR junctions and to test whether the presence of RyR1 caused altered FRET. We found that in the absence of RyR1, measureable FRET occurred between the N terminus and C terminus (residue 1636), and between the II-III loop (residue 626) and both the N and C termini; the I-II loop (residue 406) showed weak FRET with the II-III loop but not with the N terminus. Association with RyR1 caused II-III loop FRET to decrease with the C terminus and increase with the N terminus and caused I-II loop FRET to increase with both the II-III loop and N terminus. Overall, RyR1 appears to cause a substantial reorientation of the cytoplasmic α1S domains consistent with their becoming more closely packed.

Stac proteins associate with the critical domain for excitation–contraction coupling in the II–III loop of CaV1.1

In skeletal muscle, residues 720–764/5 within the CaV1.1 II–III loop form a critical domain that plays an essential role in transmitting the excitation–contraction (EC) coupling Ca2+ release signal to the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum. However, the identities of proteins that interact with the loop and its critical domain and the mechanism by which the II–III loop regulates RyR1 gating remain unknown. Recent work has shown that EC coupling in skeletal muscle of fish and mice depends on the presence of Stac3, an adaptor protein that is highly expressed only in skeletal muscle. Here, by using colocalization as an indicator of molecular interactions, we show that Stac3, as well as Stac1 and Stac2 (predominantly neuronal Stac isoforms), interact with the II–III loop of CaV1.1. Further, we find that these Stac proteins promote the functional expression of CaV1.1 in tsA201 cells and support EC coupling in Stac3-null myotubes and that Stac3 is the most effective. Coexpression in tsA201 cells reveals that Stac3 interacts only with II–III loop constructs containing the majority of the CaV1.1 critical domain residues. By coexpressing Stac3 in dysgenic (CaV1.1-null) myotubes together with CaV1 constructs whose chimeric II–III loops had previously been tested for functionality, we reveal that the ability of Stac3 to interact with them parallels the ability of these constructs to mediate skeletal type EC coupling. Based on coexpression in tsA201 cells, the interaction of Stac3 with the II–III loop critical domain does not require the presence of the PKC C1 domain in Stac3, but it does require the first of the two SH3 domains. Collectively, our results indicate that activation of RyR1 Ca2+ release by CaV1.1 depends on Stac3 being bound to critical domain residues in the II–III loop.

A shortcut to a skeletal muscle DHPR knock‐in?

Excitation–contraction (EC) coupling in mammalian skeletal muscle relies on intermolecular communication between the L-type Ca2+ channel (or 1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine receptor (RyR1). Conformational rearrangements in the DHPR that occur in response to transverse tubular membrane depolarization are transduced to RyR1 via a physical coupling between the two channels that is largely independent of any Ca2+ entry via the DHPR (reviewed in Karunasekara et al. 2009). In addition to this ‘orthograde’ signal transmitted from the DHPR to RyR1, conformational coupling also supports a ‘retrograde’ signal from RyR1 to the DHPR which increases macroscopic L-type current amplitude (Nakai et al. 1996). Although there are many other components of the macromolecular signalling complex that support this mode of communication (e.g. triadin, JP-45, Homer, FKBP12, junctophilins, DHPR γ and α2δ-1 subunits, etc.), only RyR1 and the DHPR α1S and β1a subunits are to date known to be essential for EC coupling. Mice null for any one of these three proteins die perinatally from asphyxia resulting from diaphragm paralysis. Needless to say, the inability of these neonatal mice to contract their diaphragms has precluded investigation of the basic mechanism of EC coupling in adult skeletal muscle. Fortunately, myotubes can be easily cultured from fetal or neonatal pups, and for this reason, have proven to be an exceptionally useful model system for the study of the interaction between the DHPR and RyR1. This in vitro system has enabled the expression of modified RyR1, α1S and β1a subunits in true null backgrounds, thereby facilitating identification and functional characterization of regions of these essential proteins that support communication between the DHPR and RyR1. Despite the many advantages of using null myotubes to study EC coupling, the obvious shortcoming of this system is that myotubes growing in a plastic culture dish are not differentiated muscle fibres. In particular, the fully developed triad junctions formed by the membranes of the transverse tubules and the sarcoplasmic reticulum (SR) of adult muscle are absent in cultured myotubes, which have less organized plasma membrane–SR junctions. Since conformational coupling is critically dependent on junctional ultrastructure, mechanistic differences in the DHPR–RyR1 interaction between myotubes and adult fibres may exist. In light of these potential differences, there is a need for an experimental system in which modified α1S, β1a or RyR1 clones can be expressed and evaluated in adult fibres without interference from the respective endogenous proteins. Not too long ago, Julio Vergara and colleagues introduced a technique –in vivo electroporation – in which cDNA plasmids can be delivered to an isolated set of muscles in the distal hindlimb of mature mice (see DiFranco et al. 2007). In effect, they created a transgenic system that in some respects is superior to genetically engineered mice. Specifically, expression of the plasmid of interest was limited to one or two muscles in the paw of the mouse and optimal expression was achieved in less than 2 weeks. Moreover, the technique is extremely cost-effective compared to the generation of a transgenic mouse strain. Recently, Pietri-Rouxel and colleagues (2010) similarly employed local adenoviral delivery of siRNA with the aim of ablating expression of the endogenous DHPR α1S subunit in adult tibialis anterior muscle. The attempt to create effectively dysgenic adult fibres was not entirely successful because of the inability of the siRNA to quash α1S expression completely (an inherent weakness of siRNA-based strategies), but a key role for DHPR expression in maintaining healthy muscle mass was revealed. In the study discussed below, the Vergara laboratory (DiFranco et al. 2011) has now taken the in vivo electroporation strategy to the next level; they attempt to create pharmacologically inducible α1S‘knock-in’ fibres by silencing the endogenous protein with a dihydropyridine (DHP) antagonist and replacing it with a DHP-insensitive counterpart. Their success would provide EC coupling investigators with a suitable system in which to investigate DHPR–RyR1 conformational coupling in fully differentiated muscle fibres. The first obstacle DiFranco and colleagues encountered in their shortcut to a DHPR knock-in was whether an EGFP-α1S subunit fusion construct could actually be overexpressed at reasonable level using in vivo electroporation. The Vergara laboratory has had good success expressing non-fused EGFP and an EYFP-β1a fusion construct in adult fibres (239 and 789 residues, respectively) but expression of the considerably larger EGFP-α1S construct (∼2100 residues) was by no means a trivial matter. However, much of the uncertainty concerning EGFP-α1S expression was relieved when successfully transfected flexor digitorum brevis (FDB) or interosseus (IO) fibres were identified by the fluorescence in the transverse tubules generated by the EGFP tag (DiFranco et al. 2011, Fig. 2). As one might expect with overexpression of the α1S subunit, transfected fibres had enhanced maximal gating charge movement, which indicated the presence of more DHPRs within the transverse tubule system of transfected fibres (Fig. 8). Interestingly, there was not a corresponding increase in either L-type conductance or maximal depolarization-induced SR Ca2+ release (Figs 3 and 9, respectively). The authors posited that even though there were more DHPR channels present at triad junctions in transfected fibres, there was no increase in RyR1 expression. Therefore, the ‘orphan’ DHPRs newly present in the transverse tubules were not capable of engaging EC coupling and existed in a low open probability gating state because they lacked both orthograde and retrograde coupling interactions with an RyR1 (Nakai et al. 1996). Such an explanation is quite reasonable based on previous work which has demonstrated that the retrograde influence of RyR1 substantially upregulates DHPR current density in myotubes. However, this conclusion would be considerably more sound if RyR1 expression had been tested and found to be unaltered in muscle fibres overexpressing EGFP-α1S. Having demonstrated that EGFP-α1S can be efficiently overexpressed in FDB/IO fibres, the authors overexpressed a relatively DHP-insensitive mutant α1S subunit (EGFP-α1S T935Y). The rationale for this manoeuvre was that any DHPR function (i.e. L-type Ca2+ current, voltage-sensor for SR Ca2+ release) remaining following application of a DHP antagonist could be attributed entirely to the transfected DHP-insensitive point mutant. Somewhat surprisingly, L-type current amplitude was decreased in fibres transfected with EGFP-α1S T935Y even though charge movement was increased by 44% (Fig. 8). This observation is consistent with the idea that some of the endogenous wild-type channels were displaced from the influence of RyR1 by overexpressed EGFP-α1S T935Y DHPRs that do not conduct L-type current as well as the wild-type channel (Table 2). When applied at 1 μm, the neutral DHP antagonist isradipine blocked ∼60% of the peak control L-type current in EGFP-α1S T935Y transfected fibres and ∼95% of the peak current in control fibres (naive fibres and those overexpressing EGFP-α1S, pooled; Fig. 7). From this observation, the authors concluded that the DHP-insensitive mutant was integrated into triad junctions and constituted the predominant component of the total L-type current in isradipine-treated EGFP-α1S T935Y-transfected fibres in an 8:1 ratio relative to the endogenous channel. However, the flip side of this interpretation is that the endogenous channel supported a small, but significant, portion (∼10–15%) of the DHP-insensitive current in EGFP-α1S T935Y-transfected fibres. Thus, the persistence of a contribution from the endogenous α1S subunit to DHPR function in fibres expressing EGFP-α1S T935Y is problematic for an expression system which requires a true null background. In conclusion, the work of DiFranco et al. (2011) represents a step forward in the development of an adult model for the study of conformational coupling between the DHPR and RyR1, but the current approach requires refinement to be truly useful. Perhaps species-specific sequence differences between the transfected rabbit α1S clone and the endogenous mouse α1S subunit could be exploited. If there are such regions of low conservation, then siRNA directed against the latter might be employed to eliminate the pesky isradipine-insensitive component of the native L-type current. Even though the strategy to create a pharmacologically inducible α1S knock-in fibre has not been perfected here, the in vivo electroporation strategy represents a progressive approach to the study of intrinsic muscle excitability. The great potential of the technique is the ability to overexpress or knock-down proteins that play supporting roles in EC coupling in a short period of time and at low expense in fully differentiated muscle fibres. In addition, in vivo electroporation presents an opportunity to study recessive muscle diseases such as malignant hyperthermia and central core disease by mimicking heterozygous channel expression in the muscle of afflicted individuals. For those of us who study these α1S/RyR1-linked disorders and who chase the elusive molecular underpinnings of EC coupling, the collective work of DiFranco and colleagues provides a powerful tool for these pursuits.

Stac Proteins Suppress Ca2+-Dependent Inactivation of Neuronal L-type Ca2+ Channels

Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+ channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) of l-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with β and α2-δ1 auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronal l-type Ca2+ channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channels C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI for l-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+ entry via neuronal l-type channels. SIGNIFICANCE STATEMENT Stac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle l-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry via l-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of l-type channel Ca2+ signaling in these brain regions.

Junctional trafficking and restoration of retrograde signaling by the cytoplasmic RyR1 domain

The type 1 ryanodine receptor (RyR1) in skeletal muscle is a homotetrameric protein that releases Ca2+ from the sarcoplasmic reticulum (SR) in response to an “orthograde” signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a “retrograde” signal from RyR1 increases the amplitude of the Ca2+ current produced by CaV1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RyR1. Here, we investigate whether the isolated cytoplasmic domain of RyR1 can interact structurally or functionally with CaV1.1 by producing an N-terminal construct (RyR11:4300) that lacks the C-terminal membrane domain. In CaV1.1-null (dysgenic) myotubes, RyR11:4300 is diffusely distributed, but in RyR1-null (dyspedic) myotubes it localizes in puncta at SR–PM junctions containing endogenous CaV1.1. Fluorescence recovery after photobleaching indicates that diffuse RyR11:4300 is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RyR11:4300 stably associates with PM–SR junctions. Strikingly, expression of RyR11:4300 in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca2+ current through CaV1.1, which is almost identical to the effects of full-length RyR1. Fast protein liquid chromatography indicates that ∼25% of RyR11:4300 in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RyR11:4300 is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DHPR tetrads is substantially increased by transfecting RyR-null myotubes with RyR11:4300. In summary, the RyR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR–PM junctions in myotubes only if CaV1.1 is also present and is fully functional in retrograde signaling to CaV1.1.

Association of the Alpha1S I/II Loop Peptide with Beta1A Results in Translocation of the Complex to the Cell Surface and in Clustering

The cytoplasmic domains of the skeletal muscle dihydropyridine receptor (DHPR) are involved in processes like targeting to the junctions and coupling to the ryanodine receptor (RyR1). To investigate whether defined cytoplasmic DHPR regions possess affinity for the junctions, we expressed fluorescent protein labelled variants of the α1S amino and carboxy terminus, its three loops connecting the homologous repeats, and the β1A subunit, in dysgenic (α1S null) myotubes. Individually expressed, no construct was able to recapitulate the fluorescence pattern typical of fluorescently labelled α1S correctly targeted to the junctions. Rather, the proteins exhibited a diffuse intracellular distribution. The same was true for combinatory expression of the constructs, with one exception: When the α1S loop connecting repeats I and II was co-expressed with β1A, a translocation of the two interacting proteins from the cytoplasm to the surface of the myotube occurred. Moreover, the I/II loop - β1A complex formed surface associated puncta reminiscent of fluorescently labelled junctional α1S. In fact, we could observe a partial co-localization of the complex with fluorescently labelled RyR1 when the three proteins were co-expressed in dysgenic myotubes. According to these observations, it appears possible that the I/II loop - β1A association could create a specific affinity for surface membrane regions and could thus contribute to the typical clustering of DHPRs. Additional experiments are being carried out to identify the regions within the I/II loop and within β1A required for translocation of the complex.