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Insertion of alpha1S II-III loop and C terminal sequences into alpha1H fails to restore excitation-contraction coupling in dysgenic myotubes.

Abstract：

:The L-type Ca2+ channel in skeletal muscle (alpha1S) is essential for excitation-contraction (EC) coupling. Previous studies using chimeras composed of alpha1S together with alpha1C or alpha1M demonstrated the importance of the alpha1S II-III loop and of a smaller subdomain (residues 720-764; 'ECC') in skeletal EC coupling. However, these chimeras failed to test the significance of regions outside the II-III loop, which are highly conserved between alpha1S and alpha1C. Therefore, we have injected dysgenic (alpha1S-lacking) myotubes with cDNAs encoding chimeras between alpha1S and the highly divergent T-type Ca2+ channel, alpha1H. The chimeras consisted of GFP-tagged alpha1H with one or more of the following substitutions: alpha1S II-III loop residues 720-764 ('ECC'), a putative targeting domain of the alpha1S C terminus ('target'; residues 1543-1662) or the entire alpha1S C terminus ('Cterm'; residues 1382-1873). The presence of either target or Cterm affected the expression and/or kinetics of whole-cell currents recorded from both dysgenic muscle cells and tsa-201 cells. Importantly, substitution of ECC alone into GFP-alpha1H (GFP-alpha1H + ECC), or together with either target (GFP-alpha1H + ECC + target) or Cterm (GFP-alpha1H + ECC + Cterm), was insufficient to restore electrically evoked contractions. Depolarization-induced fluorescence transients for GFP-alpha1H + ECC, GFP-alpha1H + ECC + target or GFP-alpha1H + ECC + Cterm had a bell shaped dependence upon membrane voltage (inconsistent with skeletal EC coupling) and were also exceedingly small (unlike cardiac EC coupling). The absence of EC coupling for these chimeras raises the possibility that regions of alpha1S outside of ECC and target are necessary for providing the context that allows these two domains to function in EC coupling and targeting, respectively. Additionally, an inadequate membrane density of the chimeras may have contributed to the lack of coupling.

journal_name

J Muscle Res Cell Motil

journal_title

Journal of muscle research and cell motility

authors

Wilkens CM,Beam KG

doi

10.1023/a:1024830132118

keywords:

subject

Has Abstract

pub_date

2003-01-01 00:00:00

pages

99-109

issue

eissn

0142-4319

issn

1573-2657

journal_volume

pub_type

杂志文章

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Insertion of alpha1S II-III loop and C terminal sequences into alpha1H fails to restore excitation-contraction coupling in dysgenic myotubes.