Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Polster A

PROVIDER: S-EPMC5881444 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Polster Alexander A Nelson Benjamin R BR Papadopoulos Symeon S Olson Eric N EN Beam Kurt G KG

The Journal of general physiology 20180221 4

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 i ...[more]

PMID: 29467163

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Project description:In skeletal muscle, intermolecular communication between the 1,4-dihydropyridine receptor (DHPR) and RYR1 is bidirectional: orthograde coupling (skeletal excitation-contraction coupling) is observed as depolarization-induced Ca(2+) release via RYR1, and retrograde coupling is manifested by increased L-type Ca(2+) current via DHPR. A critical domain (residues 720-765) of the DHPR alpha(1S) II-III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study, we examine the consequences of fluorescent protein insertion into different positions within the alpha(1S) II-III loop. In four constructs, a cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) tandem was introduced in place of residues 672-685 (the peptide A region). All four constructs supported efficient bidirectional coupling as determined by the measurement of L-type current and myoplasmic Ca(2+) transients. In contrast, insertion of a CFP-YFP tandem within the N-terminal portion of the critical domain (between residues 726 and 727) abolished bidirectional signaling. Bidirectional coupling was partially preserved when only a single YFP was inserted between residues 726 and 727. However, insertion of YFP near the C-terminal boundary of the critical domain (between residues 760 and 761) or in the conserved C-terminal portion of the alpha(1S) II-III loop (between residues 785 and 786) eliminated bidirectional coupling. None of the fluorescent protein insertions, even those that interfered with signaling, significantly altered membrane expression or targeting. Thus, bidirectional signaling is ablated by insertions at two different sites in the C-terminal portion of the alpha(1S) II-III loop. Significantly, our results indicate that the conserved portion of the alpha(1S) II-III loop C terminal to the critical domain plays an important role in bidirectional coupling either by conveying conformational changes to the critical domain from other regions of the DHPR or by serving as a site of interaction with other junctional proteins such as RYR1.

Dataset Information

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

Publications

Stac proteins associate with the critical domain for excitation-contraction coupling in the II-III loop of Ca<sub>V</sub>1.1.

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