Cooperation of two-domain Ca2+ channel fragments in triad targeting and restoration of excitation– contraction coupling in skeletal muscle

AUTOR(ES)

Flucher, Bernhard E.

FONTE

The National Academy of Sciences

RESUMO

The specific incorporation of the skeletal muscle voltage-dependent Ca2+ channel in the triad is a prerequisite of normal excitation–contraction (EC) coupling. Sequences involved in membrane expression and in targeting of Ca2+ channels into skeletal muscle triads have been described in different regions of the α1S subunit. Here we studied the targeting properties of two-domain α1S fragments, green fluorescent protein (GFP)-I⋅II (1–670) and III⋅IV (691–1873) expressed alone or in combination in dysgenic (α1S-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 α1S 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 Ca2+ currents and depolarization-induced Ca2+ transients, despite the severed connection between the two channel halves and the absence of amino acids 671–690 from either α1S 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 α1S 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.

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