Journal article
Ca2+-Dependent Excitation-Contraction Coupling Triggered by the Heterologous Cardiac/Brain DHPR β2a-Subunit in Skeletal Myotubes
Biophysical journal, Vol.85(6), pp.3739-3757
12/2003
DOI: 10.1016/S0006-3495(03)74790-0
PMCID: PMC1303677
PMID: 14645065
Abstract
Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR)
α
1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR
β
1a-subunit. It is unknown whether EC coupling domains present in the
β
-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
α
1S is paired with the heterologous heart/brain DHPR
β
2a-subunit. Studies were conducted in primary cultured myotubes from
β
1 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
β
2a-mediated Ca
2+
current recovered in
β
1 KO myotubes lacking the endogenous DHPR
β
1a-subunit verified formation of the
α
1S/
β
1a pair. In myotube genotypes which express no or low-density L-type Ca
2+
currents, namely
β
1 KO and RyR1 KO,
β
2a overexpression recovered a wild-type density of nifedipine-sensitive Ca
2+
currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca
2+
current recovery, there was a drastic reduction of voltage–dependent, skeletal-type EC coupling and emergence of Ca
2+
transients triggered by the Ca
2+
current. A comparison of
β
2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca
2+
-dependent Ca
2+
release triggered by the
β
2a-subunit. In
β
1 KO and RyR1 KO myotubes, the Ca
2+
-dependent EC coupling promoted by
β
2a overexpression had the following characteristics: 1), L-type Ca
2+
currents had a wild-type density; 2), Ca
2+
transients activated much slower than controls overexpressing
β
1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca
2+
current; 3), the voltage dependence of the Ca
2+
transient was bell-shaped and the maximum was centered at ∼+30 mV, consistent with the voltage dependence of the Ca
2+
current; and 4), Ca
2+
currents and Ca
2+
transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by
β
2a was inferred by the drastic reduction in maximal Ca
2+
fluorescence at large positive potentials (Δ
F
/
F
max
) in double dysgenic/
β
1 KO myotubes overexpressing the pore mutant
α
1S (E1014K) and
β
2a. The data indicate that
β
2a, upon interaction with the skeletal pore subunit
α
1S, overrides critical EC coupling determinants present in
α
1S. We propose that the
α
1S/
β
pair, and not the
α
1S-subunit alone, controls the EC coupling signal in skeletal muscle.
Details
- Title: Subtitle
- Ca2+-Dependent Excitation-Contraction Coupling Triggered by the Heterologous Cardiac/Brain DHPR β2a-Subunit in Skeletal Myotubes
- Creators
- David C Sheridan - Department of Physiology, University of Wisconsin, School of Medicine, Madison, Wisconsin 53706 USALeah Carbonneau - Department of Physiology, University of Wisconsin, School of Medicine, Madison, Wisconsin 53706 USAChris A Ahern - Department of Physiology, University of Wisconsin, School of Medicine, Madison, Wisconsin 53706 USAPriya Nataraj - Department of Physiology, University of Wisconsin, School of Medicine, Madison, Wisconsin 53706 USARoberto Coronado - Department of Physiology, University of Wisconsin, School of Medicine, Madison, Wisconsin 53706 USA
- Resource Type
- Journal article
- Publication Details
- Biophysical journal, Vol.85(6), pp.3739-3757
- Publisher
- Biophysical Society
- DOI
- 10.1016/S0006-3495(03)74790-0
- PMID
- 14645065
- PMCID
- PMC1303677
- ISSN
- 0006-3495
- eISSN
- 1542-0086
- Language
- English
- Date published
- 12/2003
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984070349802771
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