In an attempt to identify and characterize functional domains of the rabbit skeletal muscle dihydropyridine receptor ␣ 1 subunit II-III loop, we synthetized several peptides corresponding to different regions of the loop: peptides A, B, C, C1, C2, D (cf. Fig. 1 The electrical signal elicited at the T-tubule 1 membrane is transmitted to the sarcoplasmic reticulum (SR) to induce Ca 2ϩ release, which in turn leads to muscle contraction (1-8). According to the current widely accepted view, upon T-tubule depolarization a portion of the dihydropyridine receptor (DHPR), the voltage-sensing protein in the T-tubule, undergoes a conformational change to make contact with the ryanodine receptor (RyR) to open its Ca 2ϩ release channel (9 -13). The idea that the cytoplasmic loop linking Repeats II and III of the ␣ 1 subunit of the DHPR, the so-called II-III loop, may play an essential role in this process has emerged from an earlier finding that this portion of the DHPR is the critical determinant of the skeletal muscle-type Ca 2ϩ current (14). This view has been further supported by recent findings that the expressed II-III loop (both skeletal and cardiac isoforms) enhanced the ryanodine binding to the skeletal muscle RyR (15). The site important for activation of ryanodine binding was localized in the region encompassing residues Glu 666 -Glu 726 (16), which contains the phosphorylatable serine 687 (17). Furthermore, a recent study with dysgenic myotubes expressing the chimeric (skeletal/cardiac) DHPR has shown that the critical determinant of the skeletal muscle-type Ca 2ϩ transient is localized in the stretch of residues Glu 726 -Pro 742 (18). In this study, using synthetic peptides corresponding to different regions of the II-III loop of rabbit skeletal muscle DHPR ␣ 1 subunit, we identified the region responsible for triggering Ca 2ϩ release and another region for blocking the release. The implication of these findings on the E-C coupling mechanism is discussed. EXPERIMENTAL PROCEDURESPreparation-Triad-enriched microsomal fractions (triads) were prepared from rabbit back paraspinous and hind leg skeletal muscles by differential centrifugation as described previously (19). Microsomes from the final centrifugation were resuspended in a solution containing 0.3 M sucrose, 0.15 M potassium gluconate, proteolytic enzyme inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 0.8 g/ml antipain, 2.0 g/ml soybean trypsin inhibitor), 20 mM MES, pH 6.8, to a final concentration of 20 -30 mg/ml, frozen immediately in liquid N 2 , and stored at Ϫ70°C. Synthesis of II-III LoopPeptides-Peptides were synthesized on an Applied Biosystems model 431 A synthesizer employing Fmoc (N-(9-fluorenyl)methoxycarbonyl) as the ␣-amino protecting group. Peptides were cleaved and deprotected with 95% trifluoroacetic acid. Purification was carried out by reversed-phase high pressure liquid chromatography using a Rainin Instruments preparative C8 column.Ca 2ϩ Release Assays-Triads (1 mg/ml) were incubated in a solution containing 0.15 M K...
Helothermine, a protein from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to inhibit [3H]ryanodine binding to cardiac and skeletal sarcoplasmic reticulum, to block cardiac and skeletal ryanodine receptor channels incorporated into planar bilayers, and to block Ca(2+)-induced Ca2+ release triggered by photolysis of nitr-5 in saponin-permeabilized trabeculae from rat ventricle. Cloning of the helothermine cDNA revealed that the protein is composed of 223 amino acids with a molecular mass of 25,376 daltons, and apparently is stabilized by eight disulfide bridges. The peptide sequence showed significant homology with a family of cysteine-rich secretory proteins found in the male genital tract and in salivary glands. The interaction of helothermine and ryanodine receptors should serve to define functional domains within the channel structure involved in the control of Ca2+ release from sarcoplasmic reticulum.
We have previously shown that among several peptides encompassing various regions of the II-III loop of the dihydropyridine receptor alpha 1 subunit, only one peptide corresponding to the Thr671-Leu690 region (designated as peptide A) activated ryanodine binding to and induced calcium release from the sarcoplasmic reticulum [El-Hayek et al. (1995) J. Biol. Chem. 270, 22116-22118]. To further localize within peptide A the minimum unit essential for activating the sarcoplasmic reticulum calcium release channel, we synthesized variously truncated forms of peptide A and examined their ability to activate ryanodine binding. We found that the carboxy-terminal 10-residue region of peptide A encompassing Arg681-Leu690 (peptide As-10; s, skeletal muscle-type sequence) activated ryanodine binding in a RyR1-specific manner and induced calcium release even more efficiently than the 20-residue peptide A. Further truncation of one or more residue(s) of peptide As-10 virtually abolished both functions of activating ryanodine binding and inducing Ca2+ release. The activating ability of As-10 seems to be determined by at least two factors: (1) the distribution of the positively charged residues, and (2) the skeletal muscle-type amino acid sequence, as deduced from the comparison of various peptides with modified structures. These results provide evidence that the minimum essential unit for the in situ trigger of skeletal muscle excitation-contraction coupling is localized in the Arg681-Leu690 region of the II-III loop of the alpha 1 subunit of the dihydropyridine receptor.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.