Stac Proteins Research Articles

STAC proteins inhibit the voltage dependent inactivation of l-type calcium channels.

The inactivation of voltage-gated calcium channels is governed by two distinct processes: voltage dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The STAC family of adaptor proteins inhibits the CDI of L-type calcium channels (LTCC). In fact, all LTCC (CaV1.2, CaV1.3 and CaV1.4) showed reduced CDI in the presence of any STAC isoform. However, whether STAC proteins also modulate the CDI of the skeletal muscle channel CaV1.1 was initially not investigated, as CaV1.1 requires STAC3 for its functional expression. Interestingly, we could recently demonstrate that STAC3 does not inhibit the CDI, but the VDI of CaV1.1. Therefore, we hypothesized that STAC proteins inhibit also the VDI of LTCC. Indeed, when co-expressed in HEK cells, all STAC isoforms reduced the VDI of CaV1.2 and CaV1.3 currents. As for CDI, also the VDI inhibition is specific for LTCC, as the VDI of CaV2.1 and CaV2.3 is not affected by STAC proteins. As the predominant contribution to VDI is known to arise from the I-II loop interaction with the CaVβ subunit, and that STAC proteins were shown to interact with distinct cytoplasmic domains (II-III loop and C-terminus), we hypothesized that STAC proteins and membrane tethered β subunits utilize different mechanism to inhibit VDI. Indeed, co-expression of STAC3 with CaV1.3/β2a revealed additional deceleration of VDI in HEK cells. Ongoing experiments, involving STAC and channels mutants and chimeras, will reveal the molecular determinants of the VDI inhibition by STAC proteins.

STAC3 determines the slow activation kinetics of CaV 1.1 currents and inhibits its voltage-dependent inactivation.

The skeletal muscle CaV 1.1 channel functions as the voltage-sensor of excitation-contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both CaV 1.1 functional expression and EC coupling. Interestingly, STAC proteins were also reported to inhibit calcium-dependent inactivation (CDI) of L-typecalcium channels (LTCC), an important negative feedback mechanism in calcium signaling. The same could not be demonstrated for CaV 1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, CaV 1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in CaV 1.1 currents reflects the inhibitory effect of STAC3. Here, we inserted a triple mutation in the linker region of STAC3 (ETLAAA), as the analogous mutation abolished the inhibitory effect of STAC2 on CDI of CaV 1.3 currents. When coexpressed in CaV 1.1/STAC3 double knockout myotubes, the mutant STAC3-ETLAAA failed to colocalize with CaV 1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch-clamp and calcium recording experiments revealed that STAC3-ETLAAA supports CaV 1.1 functional expression and EC coupling, although at a reduced extent compared to wild-type STAC3. Importantly, STAC3-ETLAAA coexpression dramatically accelerated the kinetics of activation and inactivation of CaV 1.1 currents, suggesting that STAC3 determines the slow CaV 1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of CaV 1.1 currents, we performed patch-clamp recordings using calcium and barium as charge carriers in HEK cells. While CaV 1.1 displayed negligible CDI with STAC3, this did not increase in the presence of STAC3-ETLAAA. On the contrary, our data demonstrate that STAC3 specifically inhibits the voltage-dependent inactivation (VDI) of CaV 1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of CaV 1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC.

STAC proteins inhibit calcium- and voltage-dependent inactivation of L-type calcium channels

STAC proteins inhibit calcium- and voltage-dependent inactivation of L-type calcium channels

STAC proteins inhibit calcium and voltage dependent inactivation of L-type calcium channels

Upon depolarization, voltage-gated calcium channels (VGCC) open but then undergo inactivation. Two processes govern VGCC inactivation: calcium-dependent inactivation (CDI) and voltage-dependent inactivation (VDI). Recently, the members of the STAC family of adaptor proteins have been shown to inhibit CDI of L-type calcium channels, by binding to the C-terminal domain. Here, we demonstrate that STAC proteins also inhibit the VDI of CaV1.2 and CaV1.3. Remarkably, using a STAC mutant, we could demonstrate that the inhibition of VDI relies on the same interaction, between STAC and the calcium channel, as CDI.

Stac protein regulates release of neuropeptides.

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca2+ However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca2+ channels (CaChs) and excitation-contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca2+ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Type 8 long QT syndrome: pathogenic variants in CACNA1C-encoded Cav1.2 cluster in STAC protein binding site.

Pathogenic gain-of-function variants in CACAN1C cause type-8 long QT syndrome (LQT8). We sought to describe the electrocardiographic features in LQT8 and utilize molecular modelling to gain mechanistic insights into its genetic culprits. Rare variants in CACNA1C were identified from genetic testing laboratories. Treating physicians provided clinical information. Variant pathogenicity was independently assessed according to recent guidelines. Pathogenic (P) and likely pathogenic (LP) variants were mapped onto a 3D modelled structure of the Cav1.2 protein. Nine P/LP variants, identified in 23 patients from 19 families with non-syndromic LQTS were identified. Six variants, found in 79% of families, clustered to a 4-residue section in the cytosolic II-III loop region which forms a region capable of binding STAC SH3 domains. Therefore, variants may affect binding of SH3-domain containing proteins. Arrhythmic events occurred in similar proportions of patients with II-III loop variants and with other P/LP variants (53% vs. 48%, P = 0.41) despite shorter QTc intervals (477 ± 31 ms vs. 515 ± 37 ms, P = 0.03). A history of sudden death was reported only in families with II-III loop variants (60% vs. 0%, P = 0.03). The predominant T-wave morphology was a late peaking T wave with a steep descending limb. Exercise testing demonstrated QTc prolongation on standing and at 4 min recovery after exercise. The majority of P/LP variants in patients with CACNA1C-mediated LQT8 cluster in an SH3-binding domain of the cytosolic II-III loop. This represents a 'mutation hotspot' in LQT8. A late-peaking T wave with a steep descending limb and QT prolongation on exercise are commonly seen.

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 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.

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.

Stac Proteins Associate with the Domain of the CaV1.1 II-III Loop Critical for EC Coupling

Stac Proteins Associate with the Domain of the CaV1.1 II-III Loop Critical for EC Coupling

STAC Proteins Associate to the IQ Domain of CaV1.2 and Inhibit Calcium-Dependent Inactivation

STAC Proteins Associate to the IQ Domain of CaV1.2 and Inhibit Calcium-Dependent Inactivation

STAC proteins associate to the IQ domain of CaV1.2 and inhibit calcium-dependent inactivation

The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (CaV) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with CaV1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in CaV1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal CaV1 channels remained unknown. Using CaV1.2/2.1 chimeras expressed in dysgenic (CaV1.1-/-) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of CaV1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of CaV1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with CaV1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the CaV1.2 IQ domain with that of CaV2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the CaV1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of CaV1.2 currents.

Structural insights into binding of STAC proteins to voltage-gated calcium channels

Excitation-contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II-III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.

Stac1 and stac2 genes define discrete and distinct subsets of dorsal root ganglia neurons

Deciphering the precise in vivo function of a particular neuronal subpopulation is one of the most challenging issues in neurobiology. Dorsal root ganglia (DRG) neurons represent a powerful model system to address this fundamental question. These neurons display many morphological, anatomical and few molecular characteristics. With the aim of expanding the molecular description of the primary sensory neurons, we used Affimetrix microarrays to compare global gene expression profiles of DRG of wild type and trkA trkC/trkC knock-in mice at birth and identified several hundred potential markers of nociceptive neurons and few markers of proprioceptive neurons. Here, we describe the identification of two members of a family of putative adapter proteins STAC1 and STAC2. We found STAC1 and STAC2 being expressed in a mutually exclusive fashion in adult DRG neurons. STAC1 mainly marks peptidergic nociceptive neurons while STAC2 is expressed in a subset of nonpeptidergic nociceptors, in all trkB + neurons and in a subpopulation of proprioceptive neurons. Our expression data demonstrate that STAC proteins identify four categories of primary sensory neurons; one class of peptidergic neurons, a subset of nonpeptidergic neurons, all TrkB + neurons and a subset of proprioceptive neurons. Genetic marking of STACs-expressing sensory neurons will lend significant advance into our understanding of DRG neuronal functional diversity.