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

Abstract

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.

Figure 1

Targeting properties of the two-domain Ca²⁺ channel fragments GFP-I⋅II and III⋅IV expressed in dysgenic myotubes. Transfected myotubes were double-immunofluorescence labeled with anti-GFP to detect GFP-I⋅II, anti-α_1S to detect III⋅IV (the locations of epitopes are indicated in green in the schematic drawings of α_1S fragments below the micrographs), and with anti-RyR as independent triad marker. When coexpressed, GFP-I⋅II and III⋅IV are colocalized with one another (first column) and with the RyR1 (second column) in clusters corresponding to triad junctions (examples indicated with arrows). Individually expressed GFP-I⋅II (third column) and III⋅IV (fourth column) are not colocalized with RyR1 clusters but are expressed throughout the endoplasmic reticulum (ER)/SR system. Merged color images (bottom row) of the micrographs above show colocalization of red and green fluorescent antibodies as yellow foci and lack of colocalization as separate red and green structures. The schematic drawings show the repetitive transmembrane domain structure of the α_1S fragments expressed in the myotubes shown above. N, nuclei. (Scale bar, 20 μm.)

Figure 2

Restoration of wild-type current densities and Ca²⁺ release properties by coexpression of complementary two-domain Ca²⁺ channel fragments in dysgenic myotubes. (A) Depolarization-induced Ca²⁺ transients (upper traces) and whole-cell Ca²⁺ currents (lower traces) recorded simultaneously from α_1S-null myotubes expressing wild-type GFP-α_1S or GFP-I⋅II + III⋅IV. The holding potential was −80 mV and 200-ms test pulses to potentials between −50 and +80 mV were applied in 10-mV increments. Changes in the cytoplasmic free [Ca²⁺] were measured with Fluo-4 and shown as ΔF/F. Apparent differences in activation and inactivation kinetics of I_Ca are within the normal range of variability and are not significantly (P > 0.05) different between wild-type and GFP-I⋅II + III⋅IV. (B) Comparison of the voltage dependence of depolarization-induced Ca²⁺ transients (ΔF/F) and of peak Ca²⁺ current densities (pA/pF) recorded from α_1S-null myotubes expressing wild-type GFP-α_1S (●) and GFP-I⋅II + III⋅IV (▵). Amplitudes of transient elevations of the cytoplasmic free [Ca²⁺] and of the inward Ca²⁺ currents were identical (P > 0.05) for wild-type GFP-α_1S and coexpressed GFP-I⋅II + III⋅IV. Values represent the mean ± SEM of 9–14 recordings. The independence of Ca²⁺ transients from Ca²⁺ influx at voltages near the reversal potential (+80 mV) is characteristic of skeletal muscle EC coupling.

Figure 3

Lack of triad targeting in two-domain Ca²⁺ channel constructs containing both the β-interaction domain of the I–II connecting loop and the C-terminal triad-targeting signal. Two-domain constructs containing both of these domains were generated by fusing the C terminus of α_1S onto GFP-I⋅II (GFP-I⋅II⋅tail) or by replacing the connecting loop between repeats III and IV by that between I and II (III⋅IV⋅βin) (blue sequences in the schematic drawings of transmembrane domain structures below the micrographs). Neither GFP-I⋅II⋅tail (Left column) nor III⋅IV⋅βin (Right column) was correctly localized in triad junctions (indicated by RyR clusters) when expressed individually in dysgenic myotubes. Merged color images (bottom row) of the micrographs above show the lack of colocalization of RyR clusters and Ca²⁺ channel constructs in the ER/SR network as distinct red and green labeling patterns, respectively. N, nuclei. (Scale bar, 20 μm.)