A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D), Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta 1621-1873) or residue 1542 (Delta 1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D), For either Delta 1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced similar to 40%, and the number of functional DHPRs in the sarcolemma was reduced similar to 30%, compared with the wildtype DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by similar to 90 and similar to 55%, respectively) for h1543-1873. Fluorescence microscopy revealed that Delta 1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube, The intensity of fluorescence was greatly reduced for Delta 1543-1873, indicating a low level of protein expression, Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.