Dataset Information

STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal myotubes.

ABSTRACT: Stromal interaction molecule 1 (STIM1) along with Orai1 mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various tissues including skeletal muscle. However, the role(s) of STIM2, a homolog of STIM1, in skeletal muscle has not been well addressed. The present study, first, was focused on searching for STIM2-binding proteins from among proteins mediating skeletal muscle functions. This study used a binding assay, quadrupole time-of-flight mass spectrometry, and co-immunoprecipitation assay with bona-fide STIM2- and SERCA1a-expressing rabbit skeletal muscle. The region for amino acids from 453 to 729 of STIM2 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a). Next, oxalate-supported 45Ca2+-uptake experiments and various single-myotube Ca2+ imaging experiments using STIM2-knockdown mouse primary skeletal myotubes have suggested that STIM2 attenuates SERCA1a activity during skeletal muscle contraction, which contributes to the intracellular Ca2+ distribution between the cytosol and the SR at rest. In addition, STIM2 regulates Ca2+ movement through RyR1 during skeletal muscle contraction as well as SOCE. Therefore, via regulation of SERCA1a activity, STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal muscle, which makes it both similar to, yet different from, STIM1.

SUBMITTER: Oh MR

PROVIDER: S-EPMC5738411 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal myotubes.

Oh Mi Ri MR Lee Keon Jin KJ Huang Mei M Kim Jin Ock JO Kim Do Han DH Cho Chung-Hyun CH Lee Eun Hui EH

Scientific reports 20171220 1

Stromal interaction molecule 1 (STIM1) along with Orai1 mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various tissues including skeletal muscle. However, the role(s) of STIM2, a homolog of STIM1, in skeletal muscle has not been well addressed. The present study, first, was focused on searching for STIM2-binding proteins from among proteins mediating skeletal muscle functions. This study used a binding assay, quadr ...[more]

PMID: 29263348

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Project description:Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) alpha1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR beta1a-subunit. It is unknown whether EC coupling domains present in the beta-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit alpha1S is paired with the heterologous heart/brain DHPR beta2a-subunit. Studies were conducted in primary cultured myotubes from beta1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The beta2a-mediated Ca2+ current recovered in beta1 KO myotubes lacking the endogenous DHPR beta1a-subunit verified formation of the alpha1S/beta1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely beta1 KO and RyR1 KO, beta2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage-dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of beta2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the beta2a-subunit. In beta1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by beta2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing beta1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca2+ current; 3), the voltage dependence of the Ca2+ transient was bell-shaped and the maximum was centered at approximately +30 mV, consistent with the voltage dependence of the Ca2+ current; and 4), Ca2+ currents and Ca2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by beta2a was inferred by the drastic reduction in maximal Ca2+ fluorescence at large positive potentials (DeltaF/Fmax) in double dysgenic/beta1 KO myotubes overexpressing the pore mutant alpha1S (E1014K) and beta2a. The data indicate that beta2a, upon interaction with the skeletal pore subunit alpha1S, overrides critical EC coupling determinants present in alpha1S. We propose that the alpha1S/beta pair, and not the alpha1S-subunit alone, controls the EC coupling signal in skeletal muscle.

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Project description:We investigated the contribution of the carboxyl terminus region of the beta1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected beta1 KO myotubes were voltage clamped, and Ca(2+) transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of beta2a and a carboxyl terminus half of beta1a (beta2a 1-287/beta1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the beta1a-specific carboxyl terminus (Delta7, Delta21, Delta29, Delta35, and Delta60). Ca(2+) transients recovered by the control chimera have a sigmoidal Ca(2+) fluorescence (DeltaF/F) versus voltage curve with saturation at potentials more positive than +30 mV, independent of external Ca(2+) and stimulus duration. In contrast, the amplitude of Ca(2+) transients expressed by the truncated variants varied with the duration of the pulse, and for Delta29, Delta35, and Delta60, also varied with external Ca(2+) concentration. For Delta7 and Delta21, a 50-ms depolarization produced a sigmoidal DeltaF/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for Delta29, Delta35, and Delta60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the DeltaF/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at approximately +30 mV. The change in voltage dependence, together with the external Ca(2+) dependence and additional controls with ryanodine, indicated a loss of skeletal-type EC coupling and the emergence of an EC coupling component triggered by the Ca(2+) current. Analyses of d(DeltaF/F)/dt showed that the rate of cytosolic Ca(2+) increase during the Ca(2+) transient was fivefold faster for the control chimera than for the severely truncated variants (Delta29, Delta35, and Delta60) and was consistent with the kinetics of the DHPR Ca(2+) current. In summary, absence of the beta1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca(2+) transient, bending of the DeltaF/F versus voltage curve, and emergence of EC coupling triggered by the Ca(2+) current. The studies underscore the essential role of the carboxyl terminus region of the DHPR beta1a subunit in fast voltage dependent EC coupling in skeletal myotubes.

Dataset Information

STIM2 regulates both intracellular Ca2+ distribution and Ca2+ movement in skeletal myotubes.

Publications

STIM2 regulates both intracellular Ca<sup>2+</sup> distribution and Ca<sup>2+</sup> movement in skeletal myotubes.

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