Association of calcium channel alpha1S and beta1a subunits is required for the targeting of beta1a but not of alpha1S into skeletal muscle triads

Abstract

The skeletal muscle L-type Ca2+ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca2+ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of alpha1S-beta1a interactions in the incorporation of the functional channel complex into the triad, alpha1S and beta1a [or a beta1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. betaGFP expressed in dysgenic myotubes that lack the skeletal muscle alpha1S subunit was diffusely distributed in the cytoplasm. On coexpression with the alpha1S subunit betaGFP distribution became clustered and colocalized with alpha1S immunofluorescence. Based on the colocalization of betaGFP and alpha1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of alpha1S with and without beta1a restored Ca2+ currents and depolarization-induced Ca2+ release. The translocation of betaGFP from the cytoplasm into the junctions failed when betaGFP was coexpressed with alpha1S mutants in which the beta interaction domain had been altered (alpha1S-Y366S) or deleted (alpha1S-Delta351-380). Although alpha1S-Y366S did not associate with betaGFP it was incorporated into the junctions, and it restored Ca2+ currents and depolarization-induced Ca2+ release. Thus, beta1a requires the association with the beta interaction domain in the I-II cytoplasmic loop of alpha1S for its own incorporation into triad junctions, but stable alpha1S-beta1a association is not necessary for the targeting of alpha1S into the triads or for its normal function in Ca2+ conductance and excitation-contraction coupling.

Figure 1

Clustering of β_1a in dysgenic myotubes depends on coexpression of α_1S with intact β interaction domain. βGFP is expressed alone (a) or in combination with either the wild-type α_1S (b), the α_1S-Y366S (c), or the α_1S-Δ351–380 mutant (d). The β_1a subunit forms clusters only when coexpressed with the wild-type α_1S subunit (b). When coexpressed with the mutated α_1S subunits the distribution of βGFP is diffuse, as if expressed alone (compare c and d with a). N, myotube nuclei. (Bar, 20 μm.)

Figure 2

Differential distribution of the β_1a subunit and α_1S subunit mutants in cotransfected dysgenic myotubes. βGFP is visualized with GFP fluorescence (upper row) and α_1S subunits by immunofluorescence (lower row). a and b, wild-type α_1S is colocalized with βGFP in clusters (examples indicated with arrows). c and d, the α_1S-Y366S mutant is localized in clusters, but βGFP is diffusely distributed throughout the myotube. e and f, the α_1S-Δ351–380 mutant is localized in a dense reticular membrane system and βGFP is diffuse. The insets show a flat region of a myotube at twice the magnification. Note the tubular nature of the membrane structure containing α_1S-Δ351–380 and the clearly distinct diffuse distribution of βGFP. (Bar, 20 μm.)

Figure 3

Incorporation of α_1S wild type and α_1S-Y366S mutant into T-tubule/SR junctions. Double immunolabeling of transfected dysgenic myotubes with antibodies against the α_1S subunit (upper row) and against the RY receptor (lower row) is shown. Colocalization of the wild-type α_1S (a and b) or the α_1S-Y366S mutant (c and d) with the RY receptor in clusters is indicative of a junctional location of the two channels (examples indicated with arrows). e and f, incorporation of α_1S-Δ351–380 into junctions fails, even though the RY receptor forms normal clusters in these myotubes. Note that the RY clusters are also formed in myotubes not expressing α_1S (asterisk). N, myotube nuclei. (Bar, 20 μm.)

Figure 4

Modulation of Ca²⁺ currents by β_1a in dysgenic myotubes expressing the wild-type or Y366S mutant α_1S subunit. Patch-clamp recordings of myotubes transfected with α_1S (a) or α_1S-Y366S (b) with (β⁺) and without (β⁻) β_1a plus GFP as an expression marker are shown. The left panels show representative current traces for step depolarizations from −40 mV to +20 mV and +40 mV (asterisks). The right panels show the average I/V curves for the peak current densities (n, number of analyzed myotubes; when the current did not peak during the 1-s pulse the current value at the end of the pulse was used). In both myotubes expressing α_1S and myotubes expressing α_1S-Y366S, the peak current density is significantly (P < 0.05) increased when β_1a is coexpressed.

Figure 5

Depolarization-induced Ca²⁺ transients in dysgenic myotubes expressing the wild-type or Y366S mutant α_1S subunit. Transfected cultures were loaded with the fluorescent Ca²⁺ indicator fluo-3 AM and depolarized by passing a 2-ms, 30-V pulse (dot underneath traces) and a 2-s train of pulses at 20 Hz (bar underneath traces) across the culture chamber. Both, α_1S- and α_1S-Y366S-transfected myotubes responded with a transient increase in cytoplasmic free [Ca²⁺] to individual and tetanic stimulation. After changing to a bath solution not containing Ca²⁺, the same myotubes still responded with a [Ca²⁺] transient to depolarization (lower traces). Ca²⁺-sensitive fluorescent signals were normalized to baseline values (ΔF/F); duration of trace was 6 s.

Figure 6

Model of α_1S–β_1a interactions in the assembly of the DHP receptor complex in the skeletal muscle triad. β_1a without the α_1S subunit is localized in the cytoplasm. On coexpression β_1a and α_1S form a complex in the endoplasmic reticulum that is then incorporated into the junctional T-tubules (TT). Deletion of the β interaction domain in the I–II cytoplasmic loop blocks β_1a association and export of the α_1S mutant (α_1S-Δ) from the endoplasmic reticulum. A point mutation (α_1S-Y366S) in the high affinity β_1a binding site of α_1S (symbolized by the rectangular nipple) inhibits α_1S/β_1a complex formation but not the functional incorporation of the mutated α_1S-Y366S into the T-tubules. Dynamic interactions between free cytoplasmic β_1a and a low affinity interaction site (symbolized by the triangular notch) may modulate Ca²⁺ currents of α_1S-Y366S and may be involved in the translocation of α_1S-Y366S into the T-tubules.