Insertion of α_1S II–III loop and C terminal sequences into α_1H fails to restore excitation–contraction coupling in dysgenic myotubes

Abstract

The L-type Ca²⁺ channel in skeletal muscle (α_1S) is essential for excitation–contraction (EC) coupling. Previous studies using chimeras composed of α_1S together with α_1C or α_1M demonstrated the importance of the α_1S 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 α_1S and α_1C. Therefore, we have injected dysgenic (α_1S-lacking) myotubes with cDNAs encoding chimeras between α_1S and the highly divergent T-type Ca²⁺ channel, α_1H. The chimeras consisted of GFP-tagged α_1H with one or more of the following substitutions: α_1S II–III loop residues 720–764 (‘ECC’), a putative targeting domain of the α_1S C terminus (‘target’; residues 1543–1662) or the entire α_1S 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-α_1H (GFP-α_1H + ECC), or together with either target (GFP-α_1H + ECC + target) or Cterm (GFP-α_1H + ECC + Cterm ), was insufficient to restore electrically evoked contractions. Depolarization-induced fluorescence transients for GFP-α_1H + ECC, GFP-α_1H + ECC + target or GFP-α_1H + 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 α_1S 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.