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.     

Solubilization and isolation of dihydropyridine calcium channel blocker receptor from the rabbit skeletal muscle

A microsomal fraction of rabbit skeletal muscle was sed for the isolation of a dihydropyridine (DHP) receptor, a putative potential-dependent calcium channel. The receptor purification was followed by the binding of 3H-labeled riodipine derivative which possesses a high affinity for digitonin-solubilized DHP receptor. The DHP-Sepharose affinity chromatography of an enriched receptor fraction allowed to isolate a receptor, 60-70% homogeneous on the basis of DHP-binding activity. SDS gel electrophoresis showed that the purified receptor is composed of two subunits with molecular masses of 160 and 53 kD. The large subunit changes its electrophoretic mobility after the reduction of disulfide bonds.     

The bovine cardiac receptor for calcium channel blockers is a 195-kDa protein

The cardiac receptor for calcium channel blockers was purified from bovine microsomal membranes which contained 235 +/- 33 fmol nimodipine-binding sites/mg protein (mean +/- SEM of nine preparations). To identify the receptor during the purification 20% of its binding sites were prelabeled with (+)[3H]PN200-110. The receptor was solubilized with 0.6% digitonin and was purified to a specific density of 157 pmol/mg using a combination of ion-exchange, wheat-germ-agglutinin-Sepharose chromatography and sucrose density gradient centrifugation. In the last sucrose gradient bound (+)[3H]PN200-110 comigrated with a 195-kDa protein. ( +/-)[3H]Azidopine and [3H]ludopamil, the photoaffinity ligands for the dihydropyridine and phenylalkylamine-binding site of the calcium channel, were incorporated specifically into the 195-kDa protein. These data indicate that the bovine cardiac receptor for calcium channel blockers is a 195-kDa protein. Its molecular mass suggests that the bovine cardiac receptor differs considerably from the rabbit skeletal muscle receptor protein for calcium channel blockers.     

Purification and characterization of the dihydropyridine-sensitive voltage-dependent calcium channel from cardiac tissue

The dihydropyridine-sensitive voltage-dependent Ca2+ channel from cardiac tissue was purified 900-fold using DEAE-Sephadex A-25, concanavalin A-Sepharose, and wheat germ agglutinin-Sepharose. The purified preparation was highly enriched in a peptide of 140,000 daltons when electrophoresed on sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol, or 170,000 when electrophoresed in the presence of iodoacetamide. Polyclonal antibodies raised against the purified subunits of the rabbit skeletal muscle Ca2+ channel recognized the 170-kDa protein in preparations electrophoresed under nonreducing conditions, and the large peptide of 140 kDa and smaller peptides of 29-32 kDa in preparations analyzed under reducing conditions. Monoclonal antibodies, which were raised against the native Ca2+ channel from skeletal muscle, immunoprecipitated [3H]PN 200-110 binding activity from solubilized cardiac membranes and immunoprecipitated 125I-labeled peptides (from the purified cardiac Ca2+ channel preparation) which migrated as a single species of 170 kDa under nonreducing conditions, or as 140, 32, and 29 kDa under reducing conditions. The results show that the purified cardiac Ca2+ channel, like that previously purified from skeletal muscle, consists of a major component of 170 kDa which is comprised of a 140-kDa peptide linked by disulfide bonds to smaller peptides of 32-29 kDa. Peptide maps of the 140-kDa peptide purified from cardiac and skeletal muscle preparations were strikingly similar, suggesting a high degree of homology in their primary sequence.     

The high affinity receptor for omega-conotoxin represents calcium channels different from those sensitive to dihydropyridines in mammalian brain

A monoclonal antibody recognizing the alpha 2 delta complex of the dihydropyridine (DHP)-sensitive calcium channel of skeletal muscle immunoprecipitated most of the DHP receptor solubilized from bovine and rabbit brains, and bovine cardiac muscle. However, it did not significantly immunoprecipitate the high affinity omega-conotoxin receptor solubilized from these brains. These results indicate that the DHP receptor and the high affinity omega-conotoxin receptor are different molecules in mammalian brain.     

Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels

Partially purified fractions of dihydropyridine and phenylalkylamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalkylamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.     

Rabbit skeletal muscle microsomes contain two distinct phenylalkylamine-binding sites

Lu49888, a photoaffinity analog of verapamil, was used to identify specific binding sites for phenylalkylamines of calcium channels present in rabbit skeletal muscle microsomes. Direct binding equilibrium measurements and displacement curves of Lu49888 by its non-radioactive analog yielded an apparent single class of binding sites with Kd and Bmax values of 16.5 nM and 7.5 pmol/mg respectively. Lu49888 was specifically incorporated into three proteins of apparently 165 kDa, and 33 kDa. Incorporation into the 55-kDa protein was blocked by 10--50-fold higher concentrations of unlabeled phenylalkylamines compared to incorporation into the 165-kDa protein, suggesting that the 165-kDa and 55-kDa proteins contain a high and a low-affinity verapamil-binding site respectively. The photoaffinity-labeled proteins were solubilized by 1% digitonin or 1% Chaps in roughly equal amounts. The 165-kDa protein bound to wheat-germ-agglutinin(WGA)--Sepharose and sedimented in sucrose density gradients with the same constant as the purified dihydropyridine receptor, which has been reconstituted to a functional calcium channel. The 55-kDa membrane protein did not bind to the WGA-Sepharose column and sedimented in sucrose density gradients with a lower s value than the 165-kDa protein. The 165-kDa but not the 55-kDa membrane protein was specifically labeled by azidopine, the photoaffinity analogue of dihydropyridines. The 55-kDa protein of the purified dihydropyridine receptor was not significantly labeled by Lu49888 showing that the 55-kDa protein of the membrane is unrelated to the purified high-affinity dihydropyridine receptor.     

The 1,4-dihydropyridine receptor associated with the skeletal muscle voltage-dependent Ca2+ channel. Purification and subunit composition

The dihydropyridine receptor associated with the voltage-dependent Ca2+ channel from rabbit skeletal muscle has been purified using the tritiated derivative of (+)-PN 200-110. The drug was used not only as a marker associated with the solubilized receptor but also in direct binding experiments performed after each purification step. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of a microsomal preparation resulted in an extract with a specific binding activity of 10 pmol/mg of protein. A combination of chromatographic steps utilizing anion exchange, lectin affinity, and gel filtration resulted in an 80-fold purification to a specific binding activity of 800 pmol/mg of protein. The affinity of (+)-[3H]PN 200-110 for the solubilized receptor was only slightly altered after the purification procedure. The KD values were 0.7 and 1.8 nM on the starting material and the most purified fractions, respectively. The subunit composition of the dihydropyridine receptor was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was consistent with three polypeptides of Mr 142,000, 33,000, and 32,000. The last two small components were not covalently associated with the larger one. In spite of a careful investigation of the conditions which improved the stability of the dihydropyridine receptor, a partial denaturation could not be prevented during purification. This resulted in an underestimation of receptor purity when calculated from the maximal specific binding activity as compared to the enrichment in the three polypeptides observed after polyacrylamide gel electrophoresis. Finally, application of the same purification procedure to solubilized microsomal preparations of chick and frog skeletal muscle demonstrated the presence of a large polypeptide component of Mr 135,000-141,000 associated with the Ca2+ channel from these sources. The doublet of small molecular weight was not found with the frog muscle.     

Phosphorylation of the purified receptor for calcium channel blockers by cAMP kinase and protein kinase C

The dihydropyridine receptor purified from rabbit skeletal muscle contains three proteins of 165, 55 and 32 kDa. cAMP kinase and protein kinase C phosphorylate the 165-kDa and the 55-kDa proteins. At identical concentrations of each protein kinase, cAMP kinase phosphorylates the 165-kDa protein faster than the 55-kDa protein. Protein kinase C phosphorylates preferentially the 55-kDa protein. cAMP kinase incorporates up to 1.6 mol phosphate/mol protein into the 165-kDa protein and 1 mol/mol into the 55-kDa protein upon prolonged incubation. At a physiological concentration of cAMP kinase 1 mol phosphate is incorporated/mol 165-kDa protein within 10 min, suggesting a physiological role of this phosphorylation. Protein kinase C incorporates up to 1 mol phosphate/mol into the 55-kDa protein and less than 1 mol/mol into the 165-kDa protein. Tryptic phosphopeptide analysis reveals that cAMP kinase phosphorylates two distinct peptides in the 165-kDa protein, whereas protein kinase C phosphorylates a single peptide in the 165-kDa protein. cAMP kinase and protein kinase C phosphorylate three and two peptides in the 55-kDa protein, respectively. Mixtures of the tryptic phosphopeptides derived from the 165-kDa and 55-kDa proteins elute according to the composite of the two elution profiles. These results suggest that the 165-kDa protein, which contains the binding sites for each class of calcium channel blockers and the basic calcium-conducting structure, is a specific substrate for cAMP kinase. The 55-kDa protein apparently contains sites preferentially phosphorylated by protein kinase C.     

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.     

Photoaffinity labelling of a 33-35,000 dalton protein in cardiac, skeletal and smooth muscle membranes using a new 125I-labelled 1,4-dihydropyridine calcium channel antagonist

The binding sites for Ca2+ channel antagonists were probed using Bay P 8857 [2-iodoethyl isopropyl 1,4-dihydropyridine-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarbox ylate] that has been radiolabelled with 125I. This drug was shown to bind with high affinity to cardiac, smooth, and skeletal muscle membranes, with a KD approximately equal to 0.3 nM. A protein of molecular weight 33-35,000 daltons was specifically and irreversibly radiolabelled after irradiation of cardiac, skeletal and aortic smooth muscle membranes, incubated with the [125I]-Bay P 8857. The peptide labelled by 1,4-dihydropyridine binding therefore appears similar in size for cardiac, skeletal, and smooth muscle. This data suggests that of the three peptide subunits which reportedly comprise the skeletal and cardiac muscle 1,4-dihydropyridine receptor complex, the 33-35,000 dalton peptide contains the dihydropyridine binding site.     

A monoclonal antibody to the beta subunit of the skeletal muscle dihydropyridine receptor immunoprecipitates the brain omega-conotoxin GVIA receptor.

Antibodies against the subunits of the dihydropyridine-sensitive L-type calcium channel of skeletal muscle were tested for their ability to immunoprecipitate the high affinity (Kd = 0.13 nM) 125I-omega-conotoxin GVIA receptor from rabbit brain membranes. Monoclonal antibody VD2(1) against the beta subunit of the dihydropyridine receptor from skeletal muscle specifically immunoprecipitated up to 86% of the 125I-omega-conotoxin receptor solubilized from brain membranes whereas specific antibodies against the alpha 1, alpha 2, and gamma subunits did not precipitate the brain receptor. Purified skeletal muscle dihydropyridine receptor inhibited the immunoprecipitation of the brain omega-conotoxin receptor by monoclonal antibody VD2(1). The dihydropyridine receptor from rabbit brain membranes was also precipitated by monoclonal antibody VD2(1). However, neither the neuronal ryanodine receptor nor the sodium channel was precipitated by monoclonal antibody VD2(1). The omega-conotoxin receptor immunoprecipitated by monoclonal antibody VD2(1) showed high affinity 125I-omega-conotoxin binding, which was inhibited by unlabeled omega-contoxin and by CaCl2 but not by nitrendipine or by diltiazem. An antibody against the beta subunit of the skeletal muscle dihydropyridine receptor stained 58- and 78-kDa proteins on immunoblot of the omega-conotoxin receptor, partially purified through heparin-agarose chromatography and VD2(1)-Sepharose chromatography. These results suggest that the brain omega-conotoxin-sensitive calcium channel contains a component homologous to the beta subunit of the dihydropyridine-sensitive calcium channel of skeletal muscle and brain.     

The dihydropyridine receptor: expression of 190 kD alpha 1 subunit in crayfish muscle.

The existence of dihydropyridine receptor in crayfish striated muscle was proved by Northern blot analysis and 3H PN 200--110 binding. The alpha 1 subunit is encoded by a 8300 nt mRNA population and is expressed as 190 kD protein in crayfish T-tubular system, which binds 3H PN 200--110 (Bmax 1.5 +/- 0.4 pmol/mg protein and KD 6.2 +/- 0.8 nmol/l). The purified protein is phosphorylated by cAMP-dependent protein kinase. The dihydropyridine receptor in crayfish striated muscle also contains alpha 2 subunit, which on Northern blot gives the same signal as the alpha 2 subunit from rabbit skeletal muscle.     

Purification of a sarcoplasmic reticulum protein that binds Ca2+ and plasma lipoproteins

A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that 1) its apparent molecular weight is not changed by reduction and alkylation; 2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; 3) binding of lipoproteins is not inhibited by EDTA; and 4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins. It is unlikely that this protein ever binds lipoproteins in vivo; however, its lipoprotein binding activity has facilitated its purification to homogeneity and suggests that this protein has unusual structural features. The role of the 165-kDa protein in Ca2+ homeostasis in the sarcoplasmic reticulum, if any, remains to be determined.     

Identification of a approximately 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 approximately 170Kd polypeptide to which antibody #78 immunoblots under both reducing and non-reducing conditions.     

Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.     

Structural characterization of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel from rabbit skeletal muscle. Evidence for two distinct high molecular weight subunits

The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle triads was shown to contain four protein components of 175,000, 170,000, 52,000, and 32,000 Da when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Monoclonal antibodies capable of specifically immunoprecipitating the [3H]PN200-110-labeled dihydropyridine receptor from digitonin-solubilized triads recognized the 170,000-Da protein on nitrocellulose transfers of skeletal muscle triads, transverse tubular membranes, and purified dihydropyridine receptor. Wheat germ agglutinin peroxidase stained the 175,000-Da protein on similar nitrocellulose transfers, demonstrating that the 175,000-Da protein is the glycoprotein subunit of the purified dihydropyridine receptor. The apparent molecular weight of the Mr 170,000 protein remained unchanged with reduction, whereas the apparent molecular weight of the glycoprotein subunit shifted from 175,000 to 150,000 upon reduction. These results demonstrate that the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel from rabbit skeletal muscle contains two distinct high molecular weight subunits of 175,000 and 170,000.     

Studies on the structural requirements for the activity of the skeletal muscle dihydropyridine receptor/slow Ca2+ channel. Allosteric regulation of dihydropyridine binding in the absence of alpha 2 and beta components of the purified protein complex

A rabbit skeletal muscle dihydropyridine (DHP) receptor can be purified as an alpha 1-alpha 2-delta-beta-gamma complex, of which alpha 2 and delta are disulfide bonded. This complex has Ca2+ channel activity when incorporated into lipid bilayers. We reported recently that expression of alpha 1 in murine L cells (LCa cells) leads to appearance of both DHP binding and Ca2+ currents, and that we failed to detect alpha 2 by immunoblotting. LCa cell Ca2+ channel currents resembled those in rabbit skeletal muscle in their sensitivity to both voltage and the DHP agonist Bay K 8644, but differed in that they responded to depolarization much more slowly. We now report details of the molecular cloning of the cDNA encoding the 1857-amino acid long alpha 1 transfected into the L cells and results from studies on expression of beta, as well as, on allosteric regulation of DHP binding to these cells. The alpha 1 cDNA was cloned by a combination of cDNA library screening (5355 base pairs) and chemical synthesis (508 base pairs). Using rabbit labeled beta cDNA, which cross-reacts with murine beta mRNA, we failed to observe cross-hybridizing beta mRNA in LCa cells. Using a labeled single stranded 200-base long rabbit alpha 2 cDNA that cross-reacts with mouse alpha 2 mRNA, we likewise failed to observe cross-hybridizing alpha 2 mRNA in LCa cells and hence confirmed the absence of an endogenous murine alpha 2 in these cells. Using LCa cell membranes as DHP receptor source we found the binding of the DHP antagonist (+)-[3H]PN200-110 to be regulated by both verapamil and diltiazem as it is in rabbit skeletal muscle membranes. However, we noted a difference; at concentrations above 10(-6) M, verapamil inhibited residual DHP binding in LCa but not in skeletal muscle membranes. We conclude that neither alpha 2 nor beta are essential for expression of alpha 1 on the cell surface, or for its functioning as a voltage-gated Ca2+ channel, or for its allosteric regulation of DHP binding by Ca2+ channel antagonists. The studies neither exclude roles for gamma and delta, nor for alpha 2 or beta in determining more subtle properties of this channel.     

Characterization of DHP binding protein in crayfish striated muscle

The dihydropyridine calcium channel blocker, [3H]PN 200-110, binds specifically also to crayfish muscle membranes, though with a binding capacity smaller than that measured with rabbit or human skeletal muscle membranes. [3H]PN 200-110 binding proteins from the crayfish T-tubules were solubilized and purified on WGA Sepharose or extracted from gel. The purified protein has a molecular mass of approximately 190 kDa under nonreducing conditions and was able to transport calcium after reconstitution. Polyclonal antibodies against crayfish T-tubules enriched with purified DHP-binding protein were shown to bind to DHP-binding protein from both the crayfish and the rabbit skeletal muscle, although not with the same intensity. Electron microscopy showed the presence of ovoid particles. Our results suggest that a voltage-dependent calcium channel may be present in crayfish skeletal muscle, which is homological with the L-type calcium channel in rabbit skeletal muscle.     

Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.