Role of the alpha1 subunit of the dihydropyridine receptor in excitation-contraction coupling in skeletal myotubes.

摘要

The goal of this thesis is to unveil the molecular determinants of the DHPR alphal subunit for skeletal excitation-contraction (EC) coupling and Ca2+ current expression. Primary cultured skeletal myotubes were voltage-clamped, and Ca2+ transients were measured by confocal line scan imaging of fluo-4 fluorescence. The II-III loop of the alpha1S subunit has been deemed necessary for skeletal-type, voltage-dependent, EC coupling as well as for Ca2+ current expression. We found that functional recovery produced by the alpha1C/alpha1S II-III loop chimera is incomplete, and that presence of all tested skeletal alpha1S domains (all alpha1S inter-repeat loops, N- and C-terminus) on the alpha1C backbone, alpha1C/alpha1S all loop, was able to quantitatively recover the skeletal EC coupling phenotype. Thus, despite the importance of the II-III loop, there may be other critical determinants in alpha1S that influence EC coupling. Studies in beta1 KO skeletal myotubes revealed that the alpha1C/alpha1S all loop chimera restores a skeletal phenotype of a significantly reduced Ca2+ current expression in the absence of beta1, which can be rescued by beta1. Domains tested individually did not require beta1. We propose that the quantitative functional recovery of the alpha1C/alpha1S all loop chimera in dysgenic myotubes is realized not only by the presence of multiple skeletal alpha1S cytoplasmic domains, but also by the beta1 subunit.; We investigated the participation of the skeletal alpha1S cytoplasmic regions in RyR1-mediated retrograde enhancement of Ca2+ current expression. Previous studies have shown that the homologous alpha1S/beta1a pair does not express Ca2+ currents in dyspedic (RyR1 KO) myotubes, whereas the heterologous alpha1S/beta2a pair expresses high-density Ca 2+ currents that bypass the requirement for RyR1. Thus, characteristics of the skeletal DHPR as a weakly Ca2+-conducting DHPR voltage sensor in RyR1 KOs are derived in part by interactions with DHPR. Expression of Ca2+ current by all tested alpha1C/alpha1S chimeras as well as the alpha1C/alpha1S all loop chimera, implicates the transmembrane domains of alpha1S in retrograde signaling as well. We therefore propose that many regions within the alpha1S/beta1a pair participate in retrograde Ca2+ current enhancement by RyR1.