Skeletal Muscle Dihydropyridine Receptor Research Articles

Restoration of normal function in genetically defective myotubes by spontaneous fusion with fibroblasts

Muscular dysgenesis in mice is a genetic disease of skeletal muscle caused by the recessive mutation mdg. Muscle fibres in affected mice are paralysed because of the failure of excitation-contraction coupling. Unlike normal myotubes in primary culture, dysgenic myotubes do not contract, either spontaneously or in response to electrical stimulation. The deficiency results from mutation of the gene for the skeletal muscle dihydropyridine receptor, an essential sarcolemmal component both of excitation-contraction coupling and of the slow calcium-ion channel. It has recently been shown that the addition of fibroblasts from normal (but not dysgenic) mice to cultures of dysgenic myotubes can restore spontaneous contractions in a small fraction of these myotubes, but the mechanism for this 'rescue' was not determined. In principle, if fibroblast nuclei were able to incorporate into myotubes, such nuclei could then supply the missing muscle-specific gene product. We have now investigated this possibility using nuclear, cytoplasmic and plasmalemmal markers. We report that the rescue to contractile ability in genetically paralysed dysgenic muscle is mediated by the previously unrecognized ability of fibroblasts to fuse spontaneously with developing myotubes.

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.

Structure, Function, and Regulation of the Skeletal Muscle Dihydropyridine Receptora

Annals of the New York Academy of SciencesVolume 560, Issue 1 p. 127-137 Structure, Function, and Regulation of the Skeletal Muscle Dihydropyridine Receptora KURT G. BEAM, KURT G. BEAM Department of Physiology College of Veterinary Medicine and Biomedical Sciences Colorado State University Fort Collins, Colorado 80523Search for more papers by this authorTSUTOMU TANABE, TSUTOMU TANABE Departments of Medical Chemistry and Molecular Genetics Kyoto University Faculty of Medicine Kyoto 606, JapanSearch for more papers by this authorSHOSAKU NUMA, SHOSAKU NUMA Departments of Medical Chemistry and Molecular Genetics Kyoto University Faculty of Medicine Kyoto 606, JapanSearch for more papers by this author KURT G. BEAM, KURT G. BEAM Department of Physiology College of Veterinary Medicine and Biomedical Sciences Colorado State University Fort Collins, Colorado 80523Search for more papers by this authorTSUTOMU TANABE, TSUTOMU TANABE Departments of Medical Chemistry and Molecular Genetics Kyoto University Faculty of Medicine Kyoto 606, JapanSearch for more papers by this authorSHOSAKU NUMA, SHOSAKU NUMA Departments of Medical Chemistry and Molecular Genetics Kyoto University Faculty of Medicine Kyoto 606, JapanSearch for more papers by this author First published: June 1989 https://doi.org/10.1111/j.1749-6632.1989.tb24090.xCitations: 20 a This research was supported in part by grants from the National Institutes of Health and the Muscular Dystrophy Association (K.G.B.) and by grants from the Ministry of Education, Science and Culture of Japan, the Mitsubishi Foundation, and the Japanese Foundation of Metabolism and Diseases (S.N.). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume560, Issue1Calcium Channels: Structure and FunctionJune 1989Pages 127-137 RelatedInformation

Calcium Channels Reconstituted from the Skeletal Muscle Dihydropyridine Receptor Protein Complex and Its α1 Peptide Subunit in Lipid Bilayersa

In the first part of this study, we show that sDHPR and pDHPR preparations reconstituted into lipid bilayers formed on the tips of patch pipettes exhibit two divalent cation-selective conductance levels of 9 and 20 pS, similar in single-channel conductance to VSCC reported in a variety of intact preparations (see Pelzer et al. and Tsien et al. for review). The larger conductance level is similar to the VSCC identified in intact rat t-tubule membranes and described in sDHPR and pDHPR preparations, and shares many properties in common with activity from L-type VSCC. It is sensitive to augmentation by the DHP agonist (+/-)-BAY K 8644 and cAMP-dependent phosphorylation, and to block by the phenylalkylamine (+/-)-D600 and the inorganic blocker CoCl2. Its open-state probability and open times are increased upon depolarization as expected for a voltage-dependent activation process. Upon depolarization beyond the reversal potential, however, open-state probability and open times decline again. A reasonable way to explain the bell-shaped dependence of open times and open-state probability on membrane potential is to assume voltage-dependent ion-pore interactions that produce closing of the channel at strong negative and positive membrane potentials. By contrast, the smaller conductance level may be similar to the 10.6-pS t-tubule VSCC described by Rosenberg et al. and may best be compared with T-type VSCC. It is largely resistant to augmentation by (+/-)-BAY K 8644 and cAMP-dependent phosphorylation or block by (+/-)-D600, but is sensitive to block by CoCl2. Its open times and open-state probability show a sole dependence on membrane potential where depolarization increases both parameters sigmoidally from close to zero up to a saturating level. Both elementary conductance levels do not exhibit significant inactivation over a wide potential range, which may suggest that skeletal muscle VSCC inactivation is either poorly or not voltage-dependent at all. This possibility seems in agreement with bilayer recordings on reconstituted intact t-tubule membranes and voltage-clamp recordings on intact fibers. It supports the idea that the decline of Ca2+ current in intact skeletal muscle fibers may be due to Ca2+ depletion from the t-tubule system and/or to inactivation induced by Ca2+ release from the sarcoplasmic reticulum. We consistently observe two conductance levels of 9 and 20 pS, either singly, or together in the same bilayer from solubilized DHPR samples and even highly purified DHPR preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Ca2+ to intracellular or to extracellular sites of dihydropyridine receptor of rabbit skeletal muscle discriminates between in vitro binding of Ca2+‐channel agonist and antagonist

Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an insideout orientation. Digitonin-solubilized, purified dihydropyridine receptor is embedded in digitonin vesicles in an outside-out orientation. Ca2+ selectively stimulates binding of the Ca2+-channel antagonist [3H]PN200-110 to dihydropyridine receptor in the outside-out but not the inside-out orientation. The dissociation constant for binding Ca2+ to the extracellular Ca2+-specific binding site of dihydropyridine receptor is 2-3 microM. The data demonstrate that binding Ca2+ to the extracellular high-affinity Ca2+-binding site is required for binding dihydropyridines to dihydropyridine receptor. This binding is inhibited, however, by 1-10 mM concentrations of any divalent cation tested (Ba2+, Mn2+, Mg2+). Also, Ca2+ selectively stimulates binding of the Ca2+-channel agonist [3H]BayK8644 to dihydropyridine receptor in the inside-out orientation. The titration of this Ca2+ dependence indicates that the dissociation constant for binding Ca2+ to the intracellular Ca2+-specific binding site of dihydropyridine receptor is in the millimolar range. Thus, binding Ca2+-channel agonist or antagonist to dihydropyridine receptor is modulated by binding Ca2+ to different sites of the receptor. Measurements of dissociation rate constants for binding [3H]PN200-110 to dihydropyridine receptor in the presence of diltiazem, verapamil and/or Ca2+ indicate that Ca2+, like diltiazem or verapamil, is an allosteric effector of this receptor.

Specific Block of Calcium Channel Expression by a Fragment of Dihydropyridine Receptor cDNA

Although the structure of rabbit skeletal muscle dihydropyridine (DHP) receptor, deduced from cDNA sequence, indicates that this protein is the channel-forming subunit of voltage-dependent calcium channel (VDCC), no functional proof for this prediction has been presented. Two DNA oligonucleotides complementary to DHP-receptor RNA sequences coding for putative membrane-spanning regions of the DHP receptor specifically suppress the expression of the DHP-sensitive VDCC from rabbit and rat heart in Xenopus oocytes. However, these oligonucleotides do not suppress the expression of the DHP-insensitive VDCC and of voltage-dependent sodium and potassium channels. Thus, the gene for DHP receptor of rabbit skeletal muscle is closely related, or identical to, a gene expressed in heart that encodes a component of the DHP-sensitive VDCC. The DHP-sensitive and DHP-insensitive VDCCs are distinct molecular entities.

Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels.

Evidence from electrophysiological and ion flux studies has established that dihydropyridine-sensitive calcium channels are subject to regulation by neurotransmitter-mediated phosphorylation and dephosphorylation reactions. In the present study, we have further characterized the phosphorylation by cAMP-dependent protein kinase and a multifunctional Ca/calmodulin-dependent protein kinase of the membrane-associated form of the 165-kDa polypeptide identified as the skeletal muscle dihydropyridine receptor. The initial rates of phosphorylation of the 165-kDa peptide by both protein kinases were found to be relatively good compared to the rates of phosphorylation of established substrates of the enzymes. Phosphorylation of the 165-kDa peptide by both protein kinases was additive. Prior phosphorylation by either one of the kinases alone did not preclude phosphorylation by the second kinase. The cAMP-dependent protein kinase phosphorylated the 165-kDa peptide preferentially at serine residues, although a small amount of phosphothreonine was also formed. In contrast, after phosphorylation of the 165-kDa peptide by the Ca/calmodulin-dependent protein kinase, slightly more phosphothreonine than phosphoserine was recovered. Phosphopeptide mapping indicated that the two kinases phosphorylated the peptide at distinct as well as similar sites. Notably, one major site phosphorylated by the cAMP-dependent protein kinase was not phosphorylated by the Ca/calmodulin-dependent protein kinase, while other sites were phosphorylated to a high degree by the Ca/calmodulin-dependent protein kinase, but to a much lesser degree by the cAMP-dependent protein kinase. The results show that the 165-kDa dihydropyridine receptor from skeletal muscle can be multiply phosphorylated at distinct sites by the cAMP- and Ca/calmodulin-dependent protein kinases. As the 165-kDa peptide may be the major functional unit of the dihydropyridine-sensitive Ca channel, the results suggest that the phosphorylation-dependent modulation of Ca channel activity by neurotransmitters may involve phosphorylation of the 165-kDa peptide at multiple sites.

Identification of a ∼170K subunit of the cardiac calcium channel using a monoclonal antibody to the skeletal muscle dihydropyridine receptor

Our newly isolated monoclonal antibody (#78) specifically interacts with the 170Kd 1,4 dihydropyridine binding component of the skeletal muscle calcium channel. Dihydropyridine receptor (DHPR) from rabbit skeletal muscle and canine cardiac membranes were purified by monoclonal antibody #78 affinity chromatography. We show that DHPR from canine cardiac membranes like DHPR from rabbit skeletal membranes contain a ∼170Kd polypeptide to which antibody #78 immunoblots under both reducing and non-reducing conditions.

Identification and characterization of the dihydropyridine-binding subunit of the skeletal muscle dihydropyridine receptor.

Photoaffinity labeling of isolated triads and purified dihydropyridine receptor with [3H]azidopine and (+)-[3H]PN200-110 has been used to identify and characterize the dihydropyridine-binding subunit of the 1,4-dihydropyridine receptor of rabbit skeletal muscle. The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle triads contains four protein subunits of 175,000, 170,000, 52,000, and 32,000 Da (Leung, A., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). Photoaffinity labeling of isolated triads with [3H]azidopine resulted in specific and covalent incorporation of [3H]azidopine into only the 170,000-Da subunit of the dihydropyridine receptor and not into the 175,000-Da glycoprotein subunit of the receptor. The [3H]azidopine-labeled 170,000-Da subunit was separated from the 175,000-Da glycoprotein subunit by sequential elution from a wheat germ agglutinin-Sepharose column with 1% sodium dodecyl sulfate followed by 200 mM N-acetylglucosamine. Photoaffinity labeling of purified dihydropyridine receptor with [3H]azidopine or (+)-[3H]PN200-110 also resulted in the specific and covalent incorporation of either ligand into only the 170,000-Da subunit. Therefore, our results show that the dihydropyridine-binding subunit of the skeletal muscle 1,4-dihydropyridine receptor is the 170,000-Da subunit and not the 175,000-Da glycoprotein subunit.

Subcellular distribution and isolation of the Ca2+ antagonist receptor associated with the voltage regulated Ca2+ channel from rabbit heart muscle

The Ca2+ antagonist binding sites associated with the voltage dependent calcium channel in rabbit myocardium were found to distribute with the sarcolemmal Na+ + K+ ATPase and adenylate cyclase activities during subcellular fractionation on sucrose-density gradients. The equilibrium dissociation constants (KD) for the binding of [3H]nitrendipine and [3H]verapamil were 0.31 +/- 0.04 nM and 4.1 +/- 0.5 nM respectively, and displayed an average density of 0.55 +/- 0.05 pmol/mg and 0.4 +/- 0.03 pmol/mg protein respectively for the most enriched membrane fraction. The Ca2+ antagonist binding sites were solubilized from the membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and specific binding sites for [3H]PN200-110, [3H]verapamil and [3H]diltiazem were isolated on a wheat-germ lectin column. The binding sites for [3H]PN200-110 were enriched about 2,500 fold as compared with the original homogenate and displayed a density of 28.5 +/- 8 pmole/mg protein in the isolated fraction. Sodium dodecyl sulfate gel electrophoresis of the isolated drug binding proteins indicated enrichment of proteins of Mr 170,000, 140,000, 130,000, 100,000 and 53,000. The isolated receptor contained an intrinsic kinase activity that phosphorylated glycoproteins of Mr 170,000 and 53,000. Exogenously added cAMP-kinase stimulated phosphorylation of the 170,000, 100,000, 53,000 and 28,000 Mr glycoproteins in the receptor fraction. The results of this study indicate that the binding sites for [3H]nitrendipine, [3H]PN200-110, [3H]verapamil and [3H]diltiazem residue on glycoprotein(s) which are of sarcolemmal origin, and co-purify together on wheat germ lectin columns. The polypeptide composition of the Ca2+ antagonist binding sites from cardiac muscle appears to be very similar to that of the dihydropyridine receptor in skeletal muscle.

The 165‐KDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel

The dihydropyridine receptor purified from rabbit skeletal muscle yields in the presence of dithiothreitol and sodium dodecyl sulfate on polyacrylamide gels bands of apparent molecular mass 165 +/- 5, 130 +/- 5, 55 +/- 3, 32 +/- 2 and 28 +/- 1 kDa (chi +/- SEM, n = 12). Under nonreducing conditions, the 130 kDa and 28-kDa peptides migrate as a single peptide of 165 kDa. These peptides were separated on a HPLC size-exclusion column. The specific absorption coefficients of the isolated peptides were determined. From these a stoichiometry of 1:1.7 +/- 0.2:1.4 +/- 0.3 (chi +/- SEM of 12 experiments with three different preparations) was calculated for the 165-kDa, 55-kDa and 32-kDa peptides. The relative amount of the 130/28-kDa peptide varied with different preparations. Tryptic, chymotryptic and V-8 protease peptides of the isolated proteins suggested that the 130/28-kDa peptide was not related to the 165-kDa peptide. The dihydropyridine photoaffinity analog (+/-)-azidopine was specifically incorporated only into the 165-kDa peptide with an efficiency of about 2.4%. The azido analog of desmethoxyverapamil, LU 49888, was specifically incorporated into the same peptide with an efficiency of 1.5%. These results suggest that only the 165-kDa peptide contains the regulatory sites detected so far in the voltage-operated L-type calcium channel. They suggest further that the 130/28-kDa peptide, which migrates as a 165-kDa peptide under nonreducing conditions, does not contain high-affinity binding sites for the calcium channel blockers.

An antibody to dihydropyridine calcium entry blockers: A comparison with the calcium channel receptor in skeletal muscle

Antibodies that recognize dihydropyridine (DHP) calcium entry blockers were elicited from rabbits. A sensitive and specific radioimmunoassay for dihydropyridines was developed and its specificity compared to the DHP binding site in skeletal muscle membranes. The antibody bound [ 3H]nitrendipine with a higher affinity ( K D = 0.155 nM) than did the DHP receptor of skeletal muscle ( K D = 1–3 3 nM); however, in contrast to the DHP receptor, the antibody recognized only those DHP drugs with meta-nitrophenyl substituents at the 4-position on the DHP ring. Both the antibody and receptor exhibited stereospecificity, with each site recognizing the (+)-isomer of nicardipine as the more potent. This antibody should prove useful in our studies of some potentially irreversible DHP molecules.