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

Abstract

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 (Δ1621-1873) or residue 1542 (Δ1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Δ1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced ~40%, and the number of functional DHPRs in the sarcolemma was reduced ~30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by ~90 and ~55%, respectively) for Δ1543-1873. Fluorescence microscopy revealed that Δ1621-1873 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 Δ1543-1873, 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.