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.     

T-type Ca2+ channels in non-vascular smooth muscles

T-type Ca2+ current has been recorded in smooth muscle myocytes, and associated interstitial cells, isolated from the gastro-intestinal tract, urinary bladder, urethra, prostate gland, myometrium, vas deferens, lymphatic vessels and airways smooth muscle. By contrast, current through such channels has not been recorded from other tissues, such as the ureter. Whilst the properties of this Ca2+ current are similar in most of these cells, with respect to their voltage-dependence, ion selectivity and response to channel modulators, some differences have been recorded, most notably in the gastro-intestinal tract, and may demand a reappraisal of how a T-type Ca2+ current is characterised. The functions of such a current in different tissues remains uncertain. In most of smooth muscles discussed in this review, it is hypothesised that it underlies rhythmic or spontaneous electrical activity, especially in concert with other current-carrying systems, such as Ca2+-activated outward currents. Of equal interest is that the T-type Ca2+ channel may be a target for agents that modulate tissue function, especially in pathological conditions, or are the site of secondary effects of agents used in clinical medicine. For example, T-type Ca2+ channel modulators have been proposed to reduce overactive muscular activity in the gastro-intestinal or urinary tract, or function as tocolytic agents: and the action of volatile anaesthetics on them in airways smooth muscle requires consideration in their overall action.     

Control of voltage-dependent Ca2+ channels by G protein-coupled receptors

G proteins act as transducers between membrane receptors activated by extracellular signals and enzymatic effectors controlling the concentration of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and Ca2+. In some instances, the receptor/G protein-induced changes in the concentration of cytosolic signal molecules correlate with activity changes of voltage-dependent Ca2+ channels. Ca2+ channel modulation, in these cases, requires the participation of protein kinases whose activity is stimulated by cytosolic signal molecules. The respective protein kinases phosphorylate Ca2+ channel-forming proteins or unknown regulatory components. More recent findings suggest another membrane-confined mechanism that does not involve cytosolic signal molecules but rather a more direct control of voltage-dependent Ca2+ channels by G proteins. Modulation of Ca2+ channel activity that follows this apparently membrane-confined mechanism has been described to occur in neuronal, cardiac, and endocrine cells. The G protein involved in the hormonal stimulation of Ca2+ channels in endocrine cells may belong to the family of Gi-type G proteins, which are functionally uncoupled from activating receptors by pertussis toxin. The G protein Gs, which is activated by cholera toxin, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis toxin-sensitive G protein, possibly Go. Whether G proteins act by binding directly to Ca2+ channels or through interaction with as yet undetermined regulatory components of the plasma membrane remains to be clarified.     

Characterization of the Ca2+-switch in skeletal and cardiac muscles

To determine the significance of the global structure of the regulatory proteins in the mechanism of the Ca2+-switch in cardiac and skeletal muscle contractions, the properties of a family of Ca2+-binding proteins with 4 or 3 EF-hand motifs have been studied with desensitized skinned fiber preparations. Proteins with 4 EF hands (such as troponins C - TnCs) are dumb-bell shaped, those with 3 EF hands (parvalbumin) being ellipsoidal. The number of active sites varied between four and two. We find that the ability to anchor in the fiber is limited to proteins with 4 EF hands and, at least, two active Ca2+-binding sites, one each in the N- and C-termini. The results suggest that the dumb-bell shaped global structure is critical for the switching action in muscular contraction, and a trigger site in the N-terminus and a structural site in the C-terminus need to be active in order to regulate contractility.     

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,4-Dihydropyridine receptor associated with Ca2+ channels in human embryonic fibroblasts

By using the radioactively labeled 1,4-dihydropyridine (DHP) probe, [3H]PMD, we have demonstrated that cultured human embryonic fibroblasts grown at a low density in Eagle's medium supplemented with serum contain a single class of non-interacting DHP binding sites (Bmax, 1.2 +/- 0.3 pmol/10(6) cells; Kd, 3.9 nM). After inhibition of the DHP receptor biosynthesis by cycloheximide, the number of [3H]PMD binding sites is reduced with a half-time of 12 h, which implies a turnover rate of 30,000 +/- 7500 receptors/h per cell. With progression to confluency, the Bmax value decreased up to 0.28 +/- 0.08 pmol/10(6) cells without significant change in Kd value. When cells were grown at a low density in serum-free conditions, the number of [3H]PMD binding sites gradually increased 1.9-fold within 3 days. Addition of serum reversed this effect with the same time course. These results imply that the DHP-sensitive Ca2+ channels are involved in the control of the proliferation of human embryonic fibroblasts.     

Phosphorylation of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel by an intrinsic protein kinase in isolated triads from rabbit skeletal muscle

Isolated triads from rabbit skeletal muscle were shown to contain an intrinsic protein kinase which was neither Ca2+/calmodulin-dependent nor cAMP-dependent. The protein substrates phosphorylated by this protein kinase exhibited apparent molecular weights of 300,000, 170,000, 90,000, 80,000, 65,000, 56,000, 52,000, 51,000, 40,000, 25,000, 22,000, and 15,000. Purification of the 1,4-dihydropyridine receptor from phosphorylated triads has demonstrated that the 170,000- and 52,000-Da subunits of the 1,4-dihydropyridine receptor are phosphorylated by this intrinsic protein kinase in isolated triads. Monoclonal antibodies to the 170,000-Da subunit of the dihydropyridine receptor immunoprecipitated the 170,000-Da phosphoprotein from detergent extracts of phosphorylated triads. The mobility of the 170,000-Da phosphoprotein in sodium dodecyl sulfate-polyacrylamide gels was not changed with or without reduction, demonstrating that the 170,000-Da phosphoprotein is not the glycoprotein subunit of the receptor. Our results demonstrate that the 170,000- and 52,000-Da subunits of the dihydropyridine receptor are phosphorylated by an intrinsic protein kinase in isolated triads. In addition, our results also demonstrate that the 175,000-Da glycoprotein subunit of the dihydropyridine receptor is not phosphorylated in isolated triads by the intrinsic protein kinase, cAMP-dependent protein kinase, or endogenous Ca2+/calmodulin-dependent protein kinase.     

The effects of chronic depolarization on L-type 1,4-dihydropyridine-sensitive, voltage-dependent Ca2+ channels in chick neural retina and rat cardiac cells.

Chick neural retina cells contain functional L-type voltage-dependent Ca2+ channels sensitive to 1,4-dihydropyridines. To investigate the effects of chronic depolarization, cells were grown in medium containing elevated K+. After 4-h to 4-day treatments with elevated K+ (12-73 mM), there was a concentration-dependent decrease in high affinity [3H]PN200-110 binding. Saturation analysis of cells treated for 4 days with 40 mM K+ showed a reduction in maximum ligand binding with no change in affinity. Control and experimental Bmax values were 70.7 +/- 6.4 and 42.2 +/- 4.5 fmol/mg protein, respectively, and control and experimental KD values were 70.2 +/- 7.4 and 68.6 +/- 7.4 x 10(-12) M. The effect of chronic depolarization was time-dependent, reversible, and without effect on cellular protein content. Reduction in 45Ca2+ uptake following chronic depolarization correlated well with the reduction in [3H]PN200-110 binding. The calcium ionophore A23187, 10(-6) M for 24 h, also decreased the binding site density. The calcium channel antagonist D600 had no effect alone on [3H]PN200-110 binding; however, D600 blocked the down-regulation of calcium channels induced by chronic depolarization. The mechanism for Ca2+ channel down-regulation may involve calcium entry, since the effect was blocked by D600 and mimicked by the calcium ionophore A23187. Chronic depolarization with either elevated K+ or veratridine, or chronic treatment with A23187 had no effect on calcium channels in rat neonatal ventricular myocytes, although these cells express functional channels of the 1,4-dihydropyridine-sensitive class.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies against the 1,4-dihydropyridine receptor associated with voltage-sensitive Ca2+ channels detect similar polypeptides from a variety of tissues and species

Four monoclonal antibodies have been raised against voltage-sensitive Ca2+ channel dihydropyridine receptors from rabbit skeletal muscle. When tested by immunoblot assay of denatured transverse tubule membranes in reducing polyacrylamide gels, each recognised a single polypeptide of Mr approximately 140,000 that co-migrated with the large glycoprotein subunit of the purified receptor. In blots of nonreducing gels, a larger protein of Mr approximately 170,000 was seen and three of the antibodies recognised additional components at Mr approximately 310,000 and approximately 330,000. Crossreactive material of similar molecular mass was also seen in rabbit heart and brain, and in the skeletal muscle of other species.     

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.     

Regulation of Ca2+ release from internal stores in cardiac and skeletal muscles

It is widely accepted that Ca2+ is released from the sarcoplasmic reticulum by a specialized type of calcium channel, i.e., ryanodine receptor, by the process of Ca2+-induced Ca2+ release. This process is triggered mainly by dihydropyridine receptors, i.e., L-type (long lasting) calcium channels, directly or indirectly interacting with ryanodine receptor. In addition, multiple endogenous and exogenous compounds were found to modulate the activity of both types of calcium channels, ryanodine and dihydropyridine receptors. These compounds, by changing the Ca2+ transport activity of these channels, are able to influence intracellular Ca2+ homeostasis. As a result not only the overall Ca2+ concentration becomes affected but also spatial distribution of this ion in the cell. In cardiac and skeletal muscles the release of Ca2+ from internal stores is triggered by the same transport proteins, although by their specific isoforms. Concomitantly, heart and skeletal muscle specific regulatory mechanisms are different.     

Antibodies reveal the cytolocalization and subunit structure of the 1,4-dihydropyridine component of the neuronal Ca2+ channel

Rabbit brain synaptosomes bind the 1,4-dihydropyridine derivative (+)[3H]-PN 200-110 with an equilibrium dissociation constant of 0.04 nM and a maximal binding capacity of 400 fmol/mg of protein. Using polyclonal antibodies raised against the different components of the skeletal muscle 1,4-dihydropyridine receptor, we have demonstrated that the brain and muscle receptors share the same subunit composition comprising a large polypeptide chain of Mr 140,000 associated by disulfide bridges with a smaller peptide of Mr 32,000. These antibodies have been used in immunofluorescence staining of brain sections. They reveal a distribution of the Ca2+ channel protein similar to that of 1,4-dihydropyridine binding sites with (+)[3H]PN 200-110 by the autoradiographic technique.     

Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles

Biochemical investigation of Ca2+ release channel proteins has been carried out mainly with rabbit skeletal muscles, while frog skeletal muscles have been preferentially used for physiological investigation of Ca2+ release. In this review, we compared the properties of ryanodine receptors (RyR), Ca2+ release channel protein, in skeletal muscles between rabbit and frog. While the Ryr1 isoform is the main RyR of rabbit skeletal muscles, two isoforms, - and -RyR which are homologous to Ryr1 and Ryr3 isoforms in mammals, respectively, coexist as a homotetramer in a similar amount in frog skeletal muscles. The two isoforms in an isotonic medium show very similar property in [3H]ryanodine binding activity which is parallel to Ca2+-induced Ca2+ release (CICR) activity, and make independent contributions to the activities of the sarcoplasmic reticulum. CICR and [3H]ryanodine binding activities of rabbit and frog are qualitatively similar in stimulation by Ca2+, adenine nucleotide and caffeine, however, they showed the following quantitative differences. First, rabbit RyR showed higher Ca2+ affinity than the frog. Second, rabbit RyR showed higher activity in the presence of Ca2+ alone with less stimulation by adenine nucleotide than the frog. Third, rabbit RyR displayed less enhancement of [3H]ryanodine binding by caffeine in spite of having a similar magnitude of Ca2+ sensitization than the frog, which may explain the occasional difficulty by researchers to demonstrate caffeine contracture with mammalian skeletal muscles. Finally, but not least, rabbit RyR still showed marked inhibition of [3H]ryanodine binding in the presence of high Ca2+ concentrations in the 1 M NaCl medium, while frog RyR showed disinhibition. Other matters relevant to Ca2+ release were also discussed.     

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the Ca2+ channel structure

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.     

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.     

Unitary behavior of skeletal, cardiac, and chimeric L-type Ca2+ channels expressed in dysgenic myotubes

Skeletal and cardiac dihydropyridine receptors function both as voltage- dependent L-type calcium channels (L-channels) and as critical proteins that trigger calcium release from the sarcoplasmic reticulum in muscle. In spite of these similarities, skeletal L-channels exhibit a markedly slower activation rate than cardiac L-channels. We investigated the mechanisms underlying this difference by comparing the unitary behavior of L-channels in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, or chimeric dihydropyridine receptors. Our results demonstrate that ensemble averages activate rapidly for the purely cardiac dihydropyridine receptor and approximately five times more slowly for L-channels attributable to the purely skeletal dihydropyridine receptor or a chimeric dihydropyridine receptor in which only the first internal repeat and all of the putative intracellular loops are of skeletal origin. All of the constructs studied similarly exhibit a brief (2-ms) and a long (> or = 15-ms) open time in the presence of Bay K 8644, neither of which depend significantly on voltage. In the absence of Bay K 8644, the fraction of total open events is markedly shifted to the briefer open time without altering the rate of ensemble activation. Closed time analysis of L- channels with cardiac-like, rapid activation (recorded in the presence of dihydropyridine agonist) reveals both a brief (approximately 1-ms) closed time and a second, voltage-dependent, long-lasting closed time. The time until first opening after depolarization is three to six times faster for rapidly activating L-channels than for slowly activating L- channels and depends strongly on voltage for both types of channels. The results suggest that a voltage-dependent, closed-closed transition that is fast in cardiac L-channels and slow in skeletal L-channels can account for the difference in activation rate between these two channels.     

(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles

We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle.