Cooperation of two-domain Ca 2+ channel fragments in triad targeting and restoration of excitation– contraction coupling in skeletal muscle

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 99; no. 15; pp. 10167 - 10172
• Main Authors: Flucher, Bernhard E., Weiss, Regina G., Grabner, Manfred
• Format: Journal Article
• Language: English
• Published: 23.07.2002
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.122345799

The specific incorporation of the skeletal muscle voltage-dependent Ca 2+ channel in the triad is a prerequisite of normal excitation–contraction (EC) coupling. Sequences involved in membrane expression and in targeting of Ca 2+ channels into skeletal muscle triads have been described in different regions of the α 1S subunit. Here we studied the targeting properties of two-domain α 1S fragments, green fluorescent protein (GFP)-I⋅II (1–670) and III⋅IV (691–1873) expressed alone or in combination in dysgenic (α 1S -null) myotubes. Immunofluorescence analysis showed that GFP-I⋅II or III⋅IV expressed separately were not targeted into triads. In contrast, on coexpression the two α 1S fragments were colocalized with one another and with the ryanodine receptor in the triads. Coexpression of GFP-I⋅II and III⋅IV also fully restored Ca 2+ currents and depolarization-induced Ca 2+ transients, despite the severed connection between the two channel halves and the absence of amino acids 671–690 from either α 1S fragment. Thus, triad targeting, like the rescue of function, requires the cooperation and coassembly of the two complementary channel fragments. Transferring the C terminus of α 1S to the N-terminal two-domain fragment (GFP-I⋅II⋅tail), or transferring the I–II connecting loop containing the β interaction domain to the C-terminal fragment (III⋅IV⋅βin) did not improve the targeting properties of the individually expressed two-domain channel fragments. Thus, the cooperation of GFP-I⋅II and III⋅IV in targeting cannot be explained solely by a sequential action of the β subunit by means of the I–II loop in releasing the channel from the sarcoplasmic reticulum and of the C terminus in triad targeting.