Journal article
Truncation of the Carboxyl Terminus of the DihydropyridineReceptor [beta]1a Subunit Promotes Ca.sup.2+ Dependent Excitation-Contraction Coupling in Skeletal Myotubes
Biophysical journal, Vol.84(1), p.220
01/01/2003
DOI: 10.1016/S0006-3495(03)74844-9
PMCID: PMC1302605
PMID: 12524277
Abstract
We investigated the contribution of the carboxyl terminus region of the [beta]1a subunit of the skeletal dihydropyridine receptor (DHPR) to the mechanism of excitation-contraction (EC) coupling. cDNA-transfected [beta]1 KO myotubes were voltage clamped, and Ca.sup.2+ transients were analyzed by confocal fluo-4 fluorescence. A chimera with an amino terminus half of [beta]2a and a carboxyl terminus half of [beta]1a ([beta]2a 1-287/[beta]1a 325-524) recapitulates skeletal-type EC coupling quantitatively and was used to generate truncated variants lacking 7 to 60 residues from the [beta]1a-specific carboxyl terminus ([DELTA]7, [DELTA]21, [DELTA]29, [DELTA]35, and [DELTA]60). Ca.sup.2+ transients recovered by the control chimera have a sigmoidal Ca.sup.2+ fluorescence ([DELTA]F/F) versus voltage curve with saturation at potentials more positive than +30mV, independent of external Ca.sup.2+ and stimulus duration. In contrast, the amplitude of Ca.sup.2+ transients expressed by the truncated variants varied with the duration of the pulse, and for [DELTA]29, [DELTA]35, and [DELTA]60, also varied with external Ca.sup.2+ concentration. For [DELTA]7 and [DELTA]21, a 50-ms depolarization produced a sigmoidal [DELTA]F/F versus voltage curve with a lower than control maximum fluorescence. Moreover, for [DELTA]29, [DELTA]35, and [DELTA]60, a 200-ms depolarization increased the maximum fluorescence and changed the shape of the [DELTA]F/F versus voltage curve, from sigmoidal to bell-shaped, with a maximum at [approximately equal to]+30mV. The change in voltage dependence, together with the external Ca.sup.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.sup.2+ current. Analyses of d([DELTA]F/F)/dt showed that the rate of cytosolic Ca.sup.2+ increase during the Ca.sup.2+ transient was fivefold faster for the control chimera than for the severely truncated variants ([DELTA]29, [DELTA]35, and [DELTA]60) and was consistent with the kinetics of the DHPR Ca.sup.2+ current. In summary, absence of the [beta]1a-specific carboxyl terminus (last 29 to 60 residues of the control chimera) results in a loss of the fast component of the Ca.sup.2+ transient, bending of the [DELTA]F/F versus voltage curve, and emergence of EC coupling triggered by the Ca.sup.2+ current. The studies underscore the essential role of the carboxyl terminus region of the DHPR [beta]1a subunit in fast voltage dependent EC coupling in skeletal myotubes.
Details
- Title: Subtitle
- Truncation of the Carboxyl Terminus of the DihydropyridineReceptor [beta]1a Subunit Promotes Ca.sup.2+ Dependent Excitation-Contraction Coupling in Skeletal Myotubes
- Creators
- David C SheridanWeijun ChengChris A AhernLindsay MortensonDania AlsammaraePaola VallejoRoberto Coronado
- Resource Type
- Journal article
- Publication Details
- Biophysical journal, Vol.84(1), p.220
- DOI
- 10.1016/S0006-3495(03)74844-9
- PMID
- 12524277
- PMCID
- PMC1302605
- NLM abbreviation
- Biophys J
- ISSN
- 0006-3495
- eISSN
- 1542-0086
- Publisher
- Elsevier B.V
- Language
- English
- Date published
- 01/01/2003
- Description audience
- Academic
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984070832502771
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