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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708852114 ◽

2017 ◽

Vol 114 (45) ◽

pp. E9520-E9528 ◽

Cited By ~ 24

Author(s):

Siobhan M. Wong King Yuen ◽

Marta Campiglio ◽

Ching-Chieh Tung ◽

Bernhard E. Flucher ◽

Filip Van Petegem

Keyword(s):

Skeletal Muscle ◽

Native American ◽

Calcium Channels ◽

Sh3 Domains ◽

Mechanical Coupling ◽

Voltage Gated ◽

Ec Coupling ◽

Voltage Gated Calcium Channels ◽

Disease Variant ◽

Structural Insights

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 CaVisoforms 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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423113112 ◽

2014 ◽

Vol 112 (2) ◽

pp. 602-606 ◽

Cited By ~ 48

Author(s):

Alexander Polster ◽

Stefano Perni ◽

Hicham Bichraoui ◽

Kurt G. Beam

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Membrane Trafficking ◽

Mammalian Cells ◽

Ryanodine Receptors ◽

Adaptor Proteins ◽

Surface Expression ◽

Ec Coupling ◽

And Function

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.

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PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle

The Journal of Cell Biology ◽

10.1083/jcb.200211012 ◽

2003 ◽

Vol 160 (6) ◽

pp. 919-928 ◽

Cited By ~ 159

Author(s):

Steven Reiken ◽

Alain Lacampagne ◽

Hua Zhou ◽

Aftab Kherani ◽

Stephan E. Lehnart ◽

...

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Calcium Release ◽

Calcium Release Channel ◽

Skeletal Muscle Function ◽

Release Channel ◽

Fk506 Binding Protein ◽

Ec Coupling ◽

Increased Activity

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715997115 ◽

2018 ◽

Vol 115 (6) ◽

pp. 1376-1381 ◽

Cited By ~ 20

Author(s):

Marta Campiglio ◽

Pierre Costé de Bagneaux ◽

Nadine J. Ortner ◽

Petronel Tuluc ◽

Filip Van Petegem ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Calcium Channel ◽

Muscle Disease ◽

Excitation Contraction Coupling ◽

Contraction Coupling ◽

Calcium Dependent ◽

C Terminus ◽

Dependent Inactivation ◽

C1 Domain

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.

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Accessibility of Targeted DHPR Sites to Streptavidin and Functional Effects of Binding on EC Coupling

The Journal of General Physiology ◽

10.1085/jgp.200609730 ◽

2007 ◽

Vol 130 (4) ◽

pp. 379-388 ◽

Cited By ~ 15

Author(s):

Nancy M. Lorenzon ◽

Kurt G. Beam

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Sarcoplasmic Reticulum ◽

Conformational Changes ◽

Ec Coupling ◽

C Terminus ◽

N Terminus ◽

Electrically Evoked Contractions ◽

Evoked Contractions

In skeletal muscle, the dihydropyridine receptor (DHPR) in the plasma membrane (PM) serves as a Ca2+ channel and as the voltage sensor for excitation–contraction (EC coupling), triggering Ca2+ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) membrane. In addition to being functionally linked, these two proteins are also structurally linked to one another, but the identity of these links remains unknown. As an approach to address this issue, we have expressed DHPR α1S or β1a subunits, with a biotin acceptor domain fused to targeted sites, in myotubes null for the corresponding, endogenous DHPR subunit. After saponin permeabilization, the ∼60-kD streptavidin molecule had access to the β1a N and C termini and to the α1S N terminus and proximal II–III loop (residues 671–686). Steptavidin also had access to these sites after injection into living myotubes. However, sites of the α1S C terminus were either inaccessible or conditionally accessible in saponin- permeabilized myotubes, suggesting that these C-terminal regions may exist in conformations that are occluded by other proteins in PM/SR junction (e.g., RyR1). The binding of injected streptavidin to the β1a N or C terminus, or to the α1S N terminus, had no effect on electrically evoked contractions. By contrast, binding of streptavidin to the proximal α1S II–III loop abolished such contractions, without affecting agonist-induced Ca2+ release via RyR1. Moreover, the block of EC coupling did not appear to result from global distortion of the DHPR and supports the hypothesis that conformational changes of the α1S II–III loop are necessary for EC coupling in skeletal muscle.

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GTP gammaS removal of D-600 block of skeletal muscle excitation-contraction coupling

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.2.c572 ◽

1997 ◽

Vol 272 (2) ◽

pp. C572-C581 ◽

Cited By ~ 1

Author(s):

L. Carney-Anderson ◽

L. V. Thompson ◽

D. A. Huetteman ◽

S. K. Donaldson

Keyword(s):

Skeletal Muscle ◽

G Protein ◽

Release Mechanism ◽

Coupling Mechanism ◽

Molecular Binding ◽

Contraction Coupling ◽

Dihydropyridine Receptors ◽

Ec Coupling ◽

Continuous Bath ◽

Caffeine Contractures

G proteins interacting with dihydropyridine receptors (DHPR) in transverse tubules (TT) of skeletal muscle may have a role in skeletal excitation-contraction (EC) coupling. The aim of this study was to determine the effects of G protein-specific nucleotides [guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS) and guanosine 5'-O-(2-thiodiphosphate) (GDP betaS)] on the EC coupling mechanism in the presence of D-600, an agent that blocks EC coupling by immobilizing the voltage-sensing subunit of the DHPR in its inactivated state. By use of the mechanically peeled single-fiber preparation from rabbit adductor magnus skeletal muscle, 50 microM GTP gammaS and 500 microM GDP betaS were applied with the fiber in a D-600-induced state of blocked EC coupling. Neither nucleotide served as an independent stimulus for sarcoplasmic reticulum (SR) Ca2+ release when added to the TT polarizing bath under conditions of D-600 block. The presence of GTP gammaS or GDP betaS during a complete EC coupling cycle removed the D-600 block of EC coupling, despite continuous bath D-600. After the nucleotides were washed out, in the continued presence of D-600, the D-600 block of EC coupling was reestablished. In contrast, GTP gammaS added only during the period of TT depolarization under D-600 block did not remove the D-600 block of EC coupling, even though GTP gammaS did stimulate SR Ca2+ release. GTP gammaS had no effect on submaximum (0.5-1.0 mM) caffeine contractures and thus is unlikely to be acting through the Ca2+-induced Ca2+ release mechanism of the SR. These data suggest that the molecular binding site for GTP gammaS and GDP betaS is likely to be in the TT near the DHPR, perhaps on a G protein.

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