Journal article
A Component of Excitation-Contraction Coupling Triggered in the Absence of the T671-L690 and L720-Q765 Regions of the II-III Loop of the Dihydropyridine Receptor α1s Pore Subunit
Biophysical journal, Vol.81(6), pp.3294-3307
12/2001
DOI: 10.1016/S0006-3495(01)75963-2
PMCID: PMC1301787
PMID: 11720993
Abstract
We conducted a deletion analysis of two regions identified in the II-III loop of α1S, residues 671–690, which were shown to bind to ryanodine receptor type 1 (RyR1) and stimulate RyR1 channels in vitro, and residues 720–765 or the narrower 724–743 region, which confer excitation-contraction (EC) coupling function to chimeric dihydropyridine receptors (DHPRs). Deletion mutants were expressed in dysgenic α1S-null myotubes and analyzed by voltage-clamp and confocal fluo-4 fluorescence. Immunostaining of the mutant subunits using an N-terminus tag revealed abundant protein expression in all cases. Furthermore, the maximum recovered charge movement density was >80% of that recovered by full-length α1S in all cases. Δ671–690 had no effect on the magnitude of voltage-evoked Ca2+ transients or the L-type Ca2+ current density. In contrast, Δ720–765 or Δ724–743 abolished Ca2+ transients entirely, and L-type Ca2+ current was reduced or absent. Surprisingly, Ca2+ transients and Ca2+ currents of a moderate magnitude were recovered by the double deletion mutant Δ671–690/Δ720–765. A simple explanation for this result is that Δ720–765 induces a conformation change that disrupts EC coupling, and this conformational change is partially reverted by Δ671–690. To test for Ca2+-entry independent EC coupling, a pore mutation (E1014K) known to entirely abolish the inward Ca2+ current was introduced. α1S Δ671–690/Δ720–765/E1014K expressed Ca2+ transients with Boltzmann parameters identical to those of the Ca2+-conducting double deletion construct. The data strongly suggest that skeletal-type EC coupling is not uniquely controlled by α1S 720–765. Other regions of α1S or other DHPR subunits must therefore directly contribute to the activation of RyR1 during EC coupling
Details
- Title: Subtitle
- A Component of Excitation-Contraction Coupling Triggered in the Absence of the T671-L690 and L720-Q765 Regions of the II-III Loop of the Dihydropyridine Receptor α1s Pore Subunit
- Creators
- Chris A AhernDipankar BhattacharyaLindsay MortensonRoberto Coronado
- Resource Type
- Journal article
- Publication Details
- Biophysical journal, Vol.81(6), pp.3294-3307
- DOI
- 10.1016/S0006-3495(01)75963-2
- PMID
- 11720993
- PMCID
- PMC1301787
- ISSN
- 0006-3495
- eISSN
- 1542-0086
- Language
- English
- Date published
- 12/2001
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984070738902771
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