Cooperation of two-domain Ca(2+) channel fragments in triad targeting and restoration of excitation- contraction coupling in skeletal muscle.
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 alpha(1S) subunit. Here we studied the targeting properties of two-domain alpha(1S) fragments, green fluorescent protein (GFP)-I x II (1-670) and III x IV (691-1873) expressed alone or in combination in dysgenic (alpha(1S)-null) myotubes. Immunofluorescence analysis showed that GFP-I x II or III x IV expressed separately were not targeted into triads. In contrast, on coexpression the two alpha(1S) fragments were colocalized with one another and with the ryanodine receptor in the triads. Coexpression of GFP-I x II and III x 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 alpha(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 alpha(1S) to the N-terminal two-domain fragment (GFP-I x II x tail), or transferring the I-II connecting loop containing the beta interaction domain to the C-terminal fragment (III x IV x beta in) 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 beta subunit by means of the I-II loop in releasing the channel from the sarcoplasmic reticulum and of the C terminus in triad targeting.
Department of Physiology, University of Innsbruck, A-6020 Innsbruck, Austria. bernard.e.flucher@uibk.ac.at
Bernhard E
BE
Flucher
Regina G
RG
Weiss
Manfred
M
Grabner
D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000502:physiology*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D015640:Ion Channel Gating / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology*; D009126:Muscle Relaxation / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology*; D010446:Peptide Fragments / Q000502:physiology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D020746:Calcium Channels, L-Type; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins