FIGURE 4.

Interaction between the 4,5-loop and the C-tail. A, putative sequences of the highly conserved loop between transmembrane domains 4 and 5 from all three IP₃R isoforms and RyR1 are shown. The indicated sequences for IP₃R1 and RyR1 were expressed as GST fusion proteins. B, GST alone or GST-IP₃R1 loop and GST-RyR1 loop were immobilized on GSH-agarose and incubated with bacterial lysate expressing the C-tail as described under “Experimental Procedures.” Bound C-tail was detected with CT-1 Ab. The input lane was 10 μg of the C-tail lysate. The blots were stripped and reprobed with anti-GST Ab. The data from three experiments were quantitated. C, binding of immobilized GST-IP₃R1 loop to bacterial lysates expressing wild-type and mutant C-tails was carried out as described for B. The amount of C-tail bound was estimated by immunoblotting with CT-1 Ab after normalization for differences in expression levels of the constructs determined by analyzing 10-μg aliquots of the input lysates. The data shown are the means ± S.E. of three experiments. D, bacterial lysates expressing His₆-tagged wild-type or mutant receptors were partially purified on Talon resin as described under “Experimental Procedures.” Aliquots (3 μg of protein) were incubated for 30 min in a final volume of 50 μl with 125 μm of the noncleavable cross-linker SMPH. The reaction was terminated by the addition of 5 mm Tris-HCl and SDS sample buffer. The reaction was run on 17.5% gels, and cross-linked bands were detected with CT-1 Ab. At lower exposure, the cross-linked bands appear as a closely spaced doublet.