Figure 4.
Characterization of recombinant RyR2 amino-terminal domains. (A) Cytoplasmic (left panel) and microsomal (right panel) fractions (100 μg) obtained from CHORxR cells expressing RyR2 N-terminal constructs were immunoblotted using pAb2143. Full-length recombinant RyR2 was used as control (MW ∼565 kDa). (B) Intracellular Ca2+ in CHORxR cells expressing RyR2 N-terminal domains was determined in resting cells and after the addition of caffeine (10 mM, top traces) and 4-CMC (1 mM, bottom traces; arrowed). Bar, 10 s. (C) The localization of RyR2 domains in CHORxR or G7 and E6 cells induced with ponA (5 μM) was determined using confocal microscopy. Coincidence of eGFP-tagged RyR2 TM domains (green) and dsRed tagged N-terminal domain (red) appears yellow. Bar, 10 μm. (D) Analysis of coincident staining between eGFP- and dsRed-tagged RyR2 constructs is expressed as the percentage of total cellular fluorescent signal of the RyR TM domains (green) that could be directly over-laid with fluorescence signal from dsRed tagged fusion proteins at 1024 × 1024 pixel resolution. G7 and E6 cells are represented as white and black bars, respectively. *p < 0.005 when compared with cells expressing untagged dsRed (n = 4, >12 cells per experiment).