Bidirectional signaling between calcium channels of skeletal muscle requires multiple direct and indirect interactions

Sheridan et al. 10.1073/pnas.0609473103.

Supporting Information

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Methods

Fig. 6. Myotubes expressing Ch-4 construct present either small arrays of tetrads within a larger cluster, as shown here, or clusters of particles that are randomly dispersed (data not shown). On the average (Table 2), the content of tetrads in the clusters was lower than for RyR1 but higher than for RyR3. Chimeras RyR3+D2, Ch-4, Ch-2rev, and RyR1-D2 do not restore tetrad arrays; they form clusters of randomly dispersed DHPR particles with rare (apparent) tetrads. RyR1-D2 made small clusters, but the particle content and density in each cluster (see Table 2) would have been sufficient to form tetrads, if allowed by the construct.

Fig. 7. Reduced EC coupling efficiency is not the result of failed functional targeting of chimeric RyR1/RyR3 constructs. Subcellular localization of chimeric receptors transiently expressed in 1B5 myotubes reveals that RyRs are localized in clusters at the cell periphery that correspond to the junctions of the SR with the plasma membrane. (A-C, E, and F) Note that chimeric constructs that restore EC coupling with high (B and C) or medium (A) efficiency presented the same immunolabeling pattern that those that fail to rescue any coupling (E and F). (D) Ch-4, however, showed the highest variation in targeting: Some cells had very small clusters of RyRs at the cell periphery, whereas others (as shown here) had larger clusters.

Fig. 8. Histograms display the number of cells (frequency) that produced a given peak Ca²⁺ current distribution range by each construct as expressed in primary culture dyspedic myotubes. The bins were 0.1-3, 3.1-6, 6.1-9, and >9 pA/pF. Dashed vertical lines denote the separation between small, dyspedic-like currents, 0.1-3 pA/pF, and those >3.1 pA/pF.

Supporting Methods

Immunolabeling. Cultured cells were fixed in methanol for a minimum of 20 min at
-20°C, blocked in PBS (26.7 mM Na₂HPO₄/7.3 mM KH₂PO₄/136.8 mM NaCl/2.6 mM KCl) containing 1% BSA and 10% goat serum for 1 h, incubated first in antibody 34°C (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City) and then with secondary antibody (Cy3 conjugated; Jackson ImmunoResearch Laboratories, Lexington, KY) for 2 h at room temperature. The specimens were observed using either fluorescent microscope (Olympus IX70, Center Valley, PA) or a confocal laser-scanning microscope (LSM510, Carl Zeiss, Germany) using a ´63 oil immersion lens.

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