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 ß 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 channel's 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.

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

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 Ca(2+) release from the SR and retrograde coupling by which RyR1 increases the magnitude of the Ca(2+) 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 ß1a and α2-δ1 subunits) and the generation of large Ca(2+) currents, we found that without Stac3 the auxiliary γ1 subunit also supported membrane expression of CaV1.1/ß1a/α2-δ1, but that this combination generated only tiny Ca(2+) 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 Ca(2+) currents or EC coupling Ca(2+) release. Expression of WT Stac3 in Stac3 KO myotubes fully restored Ca(2+) currents and EC coupling Ca(2+) release, whereas expression of Stac3W280S (containing the Native American myopathy mutation) partially restored Ca(2+) 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.

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

Excitation-contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

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) α(1S) subunit.

The skeletal muscle dihydropyridine receptor (DHPR) in the t-tubular membrane serves as the Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling, triggering Ca(2+) release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). The two proteins appear to be physically linked, and both the α(1S) and ß(1a) subunits of the DHPR are essential for EC coupling. Within α(1S), cytoplasmic domains of importance include the I-II loop (to which ß(1a) 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 α(1S) cytoplasmic domains. Expression of these constructs in dyspedic (RyR1 null) and dysgenic (α(1S) 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 α(1S) domains consistent with their becoming more closely packed.

Lepidium as a model system for studying the evolution of fruit development in Brassicaceae.

Fruits represent a key innovation of the flowering plants that facilitates seed dispersal. In many species of the plant family Brassicaceae dehiscent fruits develop in which seed dispersal occurs through a process termed 'pod-shatter'. In the case of dehiscence, the fruit opens during fruit maturation. Phylogeny reconstructions using molecular markers indicate that the development of dehiscent fruits is the ancestral condition within the genus Lepidium s.l., but that indehiscent fruits evolved independently several times from dehiscent fruits. With Lepidium campestre and Cardaria pubescens (also known as Lepidium appelianum), very closely related taxa with dehiscent and indehiscent fruits, respectively, were identified which constitute a well-suited model system to determine the molecular genetic basis of evolutionary changes in fruit dehiscence. Following the rationale of evolutionary developmental biology ('evo-devo') phylomimicking mutants with indehiscent fruits of the close relative Arabidopsis have been used to define the candidate genes ALC, FUL, IND, RPL, and SHP1/2 which might be involved in the origin of indehiscent fruits in Cardaria. Comparative expression studies in L. campestre and C. pubescens are used to identify differentially expressed genes and thus to narrow down the number of candidate genes. Reciprocal heterologous transformation experiments may help us to distinguish direct from indirect developmental genetic causes of fruit indehiscence, and to assess the contribution of cis- and trans-regulatory changes.

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.

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.

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 (Ca(V)1.2) 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 (Ca(V)1.1). However, there are several Ca(V)1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding Ca(V)1.2 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 Ca(V)1.1 and Ca(V)1.2. We found that in the presence of Ca(2+) (either millimolar or that in resting myotubes), CaM bound strongly to C termini of Ca(V)1.2 but not of Ca(V)1.1. Furthermore, replacement of two residues (Tyr(1657) and Lys(1662)) within the IQ motif of a C-terminal Ca(V)1.2 construct with the divergent residues of Ca(V)1.1 (His(1532) and Met(1537)) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in Ca(V)1.1 caused a gain of CaM binding. In full-length Ca(V)1.2, substitution of these same two divergent residues with those of Ca(V)1.1 (Y1657H, K1662M) eliminated calcium-dependent inactivation of the heterologously expressed channel. Thus, our results reveal that a conserved difference between the IQ motifs of Ca(V)1.2 and Ca(V)1.1 has a profound effect on both CaM binding and calcium-dependent inactivation.