Localization in the II-III Loop of the Dihydropyridine Receptor of a Sequence Critical for Excitation-Contraction Coupling

Abstract

Skeletal and cardiac muscles express distinct isoforms of the dihydropyridine receptor (DHPR), a type of voltage-gated Ca2؉ channel that is important for excitation-contraction (EC) coupling. However, entry of Ca 2؉ through the channel is not required for skeletal muscletype EC coupling. Previous work (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature 346, 567-569) revealed that the loop between repeats II and III (II-III loop) is an important determinant of skeletal-type EC coupling. In the present study we have further dissected the regions of the II-III loop critical for skeletal-type EC coupling by expression of cDNA constructs in dysgenic myotubes. Because Ser 687 of the skeletal II-III loop has been reported to be rapidly phosphorylated in vitro, we substituted this serine with alanine, the corresponding cardiac residue. This alaninesubstituted skeletal DHPR retained the ability to mediate skeletal-type EC coupling. Weak skeletal-type EC coupling was produced by a chimeric DHPR, which was entirely cardiac except for a small amount of skeletal sequence (residues 725-742) in the II-III loop. Skeletal-type coupling was stronger when both residues 725-742 and adjacent residues were skeletal (e.g. a chimera containing skeletal residues 711-765). However, residues 725-742 appeared to be critical because skeletaltype coupling was not produced either by a chimera with skeletal residues 711-732 or by one with skeletal residues 734 -765. Dihydropyridine receptors (DHPRs)1 in skeletal and cardiac muscle are closely related proteins encoded by two different genes (1, 2). In both muscle types, DHPRs serve dual functions (2-6) as voltage-gated, L-type Ca 2ϩ channels and as a trigger for excitation-contraction (EC) coupling, which controls the release of Ca 2ϩ through ryanodine receptors (RyRs) (7) in the sarcoplasmic reticulum. However, the mechanism of EC coupling is different in cardiac and skeletal muscle. In cardiac muscle, depolarization causes opening of L-type Ca 2ϩ channels, and the resulting entry of extracellular Ca 2ϩ triggers RyRs to release Ca 2ϩ (6). In skeletal muscle, EC coupling does not require Ca 2ϩ entry (8); rather, depolarization causes some other kind of signal to be transmitted from DHPRs to RyRs. To identify regions that are critical for this skeletal-type EC coupling, we have previously used the approach of constructing cDNAs encoding chimeras of the skeletal and cardiac DHPRs (3). Expression of these chimeric DHPRs in dysgenic myotubes, which lack a functional gene for the skeletal DHPR (9), revealed that the putative cytoplasmic region between repeats II and III (II-III loop, amino acids 666 -791) of the skeletal DHPR is an important determinant of skeletal-type EC coupling (3).Comparison of the skeletal and cardiac DHPRs reveals differences scattered throughout the II-III loops. One important difference is the presence of a site (Ser 687 ) in the skeletal II-III loop, which is phosphorylated by cyclic AMP-dependent protein kinase (PKA) (1, 10) and is lacking in t...

Involvement of the Glu724–Pro760 Region of the Dihydropyridine Receptor II-III Loop in Skeletal Muscle-type Excitation-Contraction Coupling

“…The [peptide C] dependence of inhibition of depolarization-induced conformational change was similar to that of peptide A-induced conformational change, supporting the notion that voltage-dependent activation of the RyR is mediated by the region of the II-III loop corresponding to peptide A. Third, replacement of the portion of peptide C, which corresponds to the determinant of skeletal muscle-type E-C coupling (14), from the skeletal muscle-type sequence to the cardiac type produced a significant reduction of the blocking ability of peptide C. This suggests that skeletal-type E-C coupling requires not only the activating function localized in the peptide A region of the II-III loop but also the blocking function residing in the peptide C region. Based upon these findings, we propose the following mechanism (cf.…”

“…We also carried out the same type of experiment with some control peptides. As described previously (12) (14) described that there is a considerable amount of inconsistency in the literature concerning the proposed locations of critical domain(s) of the II-III loop. However, many (if not all) of those apparently inconsistent findings, together with the present findings, may be explained by a general hypothesis as elaborated below.…”

“…However, many (if not all) of those apparently inconsistent findings, together with the present findings, may be explained by a general hypothesis as elaborated below. First, the finding by Nakai et al (14) that replacement of a short segment of the cardiac loop with the corresponding skeletal residues Phe 725 -Pro 742 was sufficient to produce skeletal type E-C coupling has led to their suggestion that this region may serve as an "agonist," rather than the peptide A region being the activator. This suggestion was based upon the assumption that the cardiac sequence of the peptide A region of the loop would have no capability of activating the RyR1 (14).…”

“…First, the finding by Nakai et al (14) that replacement of a short segment of the cardiac loop with the corresponding skeletal residues Phe 725 -Pro 742 was sufficient to produce skeletal type E-C coupling has led to their suggestion that this region may serve as an "agonist," rather than the peptide A region being the activator. This suggestion was based upon the assumption that the cardiac sequence of the peptide A region of the loop would have no capability of activating the RyR1 (14). As a matter of fact, as shown in our recent report, both skeletal and cardiac sequences of the critical 10-residue portion of peptide A (peptide A-10) can activate the RyR1, although cardiac peptide A-10 is somewhat weaker than its skeletal counterpart (cf.…”

“…This suggests that the Glu 724 -Pro 760 region of the loop may serve as an antagonist of the activator described above. The idea that this portion is critical for E-C coupling has also emerged from the recent report by Nakai et al (14). According to the report, chimeric replacement of the Phe 725 -Pro 742 region of the II-III loop (the region corresponding to the N-terminal half of peptide C) from cardiac type to skeletal muscle type conferred the E-C coupling properties of skeletal muscle-type to dysgenic myotubes expressing cardiac-type DHP receptor.…”