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1.
Sulfhydryl oxidation modifies the calcium dependence of ryanodine-sensitive calcium channels of excitable cells. 总被引:10,自引:1,他引:9
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The calcium dependence of ryanodine-sensitive single calcium channels was studied after fusing with planar lipid bilayers sarcoendoplasmic reticulum vesicles isolated from excitable tissues. Native channels from mammalian or amphibian skeletal muscle displayed three different calcium dependencies, cardiac (C), mammalian skeletal (MS), and low fractional open times (low Po), as reported for channels from brain cortex. Native channels from cardiac muscle presented only the MS and C dependencies. Channels with the MS or low Po behaviors showed bell-shaped calcium dependencies, but the latter had fractional open times of <0.1 at all [Ca2+]. Channels with C calcium dependence were activated by [Ca2+] < 10 microM and were not inhibited by increasing cis [Ca2+] up to 0.5 mM. After oxidation with 2,2'-dithiodipyridine or thimerosal, channels with low Po or MS dependencies increased their activity. These channels modified their calcium dependencies sequentially, from low Po to MS and C, or from MS to C. Reduction with glutathione of channels with C dependence (native or oxidized) decreased their fractional open times in 0.5 mM cis [Ca2+], from near unity to 0.1-0.3. These results show that all native channels displayed at least two calcium dependencies regardless of their origin, and that these changed after treatment with redox reagents. 相似文献
2.
The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub microM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed. 相似文献
3.
Dihydropyridine-sensitive calcium channels in cardiac and skeletal muscle membranes: studies with antibodies against the alpha subunits 总被引:2,自引:0,他引:2
Polyclonal antibodies (PAC-2) against the purified skeletal muscle calcium channel were prepared and shown to be directed against alpha subunits of this protein by immunoblotting and immunoprecipitation. These polypeptides have an apparent molecular weight of 162,000 without reduction of disulfide bonds. Under conditions where the functional properties of the purified skeletal muscle calcium channel are retained, beta subunits (Mr 50,000) and gamma subunits (Mr 33,000) are coprecipitated, demonstrating specific noncovalent association of these three polypeptides in the purified skeletal muscle channel. PAC-2 immunoprecipitated cardiac calcium channels labeled with [3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- (methoxycarbonyl)pyridine-3-carboxylate ([3H]PN200-110) at a 3-fold higher concentration than skeletal muscle channels. Preincubation with cardiac calcium channels blocked only 49% of the immunoreactivity of PAC-2 toward skeletal muscle channels, indicating that these two proteins have both homologous and distinct epitopes. The immunoreactive component of the cardiac calcium channel was identified by immunoprecipitation and polyacrylamide gel electrophoresis as a polypeptide with an apparent molecular weight of 170,000 before reduction of disulfide bonds and 141,000 after reduction, in close analogy with the properties of the alpha 2 subunits of the skeletal muscle channel. It is concluded that these two calcium channels have a homologous, but distinct, alpha subunit as a major polypeptide component. 相似文献
4.
M K Ahlijanian J Striessnig W A Catterall 《The Journal of biological chemistry》1991,266(30):20192-20197
Antibodies that recognize the alpha 2 delta and alpha 1 subunits of skeletal muscle L-type calcium channels have been used to investigate the subunit components and phosphorylation of omega-conotoxin (omega-CgTx)-sensitive N-type calcium channels from rabbit brain. Photolabeling of the N-type channel with a photoreactive derivative of 125I-omega-CgTx results in the identification of a single polypeptide of 240 kDa. MANC-1, a monoclonal antibody recognizing alpha 2 delta subunits of L-type calcium channels from skeletal muscle, immunoprecipitates the omega-CgTx-labeled 240-kDa polypeptide and approximately 6% of the digitonin-solubilized 125I-omega-CgTx-labeled N-type channels. MANC-1 also immunoprecipitates a phosphoprotein of 240 kDa that comigrates with 125I-omega-CgTx-labeled N-type calcium channels, but not with L-type calcium channels, in sucrose gradients. Both cAMP-dependent protein kinase and protein kinase C are effective in the phosphorylation of this polypeptide. Similar to the alpha 1 subunits of skeletal muscle L-type calcium channels, the immunoprecipitation of the 240-kDa phosphoprotein by MANC-1 is prevented by the detergent Triton X-100. Anti-CP-(1382-1400), an antipeptide antibody against a highly conserved segment of the alpha 1 subunits of calcium channels, immunoprecipitates the 240-kDa phosphopeptide in Triton X-100. The 240-kDa protein is phosphorylated to a stoichiometry of approximately 1 mol of phosphate/mol of omega-CgTx-binding N-type calcium channels by both cAMP-dependent protein kinase and protein kinase C. Our results show that the 240-kDa polypeptide is an alpha 1-like subunit of an omega-CgTx-sensitive N-type calcium channel. The N-type calcium channels containing this subunit are phosphorylated by cAMP-dependent protein kinase and protein kinase C and contain noncovalently associated alpha 1-like and alpha 2 delta-like subunits as part of their oligomeric structure. 相似文献
5.
Molecular diversity of L-type calcium channels. Evidence for alternative splicing of the transcripts of three non-allelic genes 总被引:22,自引:0,他引:22
E Perez-Reyes X Y Wei A Castellano L Birnbaumer 《The Journal of biological chemistry》1990,265(33):20430-20436
The diversity of L-type calcium channels was probed using the polymerase chain reaction and primers based on regions conserved in the L-type skeletal muscle (CaCh 1) and cardiac calcium channels (CaCh 2). Related sequences were amplified from human heart, hamster heart, rabbit heart, mouse ovary, mouse BC3H1 cells, and hamster insulin-secreting (HIT) cells. Sequencing of various clones revealed the presence of alternate splicing in gene products coding for CaCh 1, CaCh 2, and a related calcium channel. This related gene product, which we refer to as neuroendocrine or CaCh 3, is expressed in brain and endocrine cells. The diverse products can be explained by the use of alternate exons of equal size, which account for changes in amino acid composition, in combination with an alternate splice acceptor site or an exon skipping event, which produces channels of variable length. Four variants were defined for the gene 3 product, subtypes 3a, 3b, 3c, and 3d that differed in both the sequence of the third membrane spanning segment of the fourth repeat unit (IVS3) and in the size of the linker between this and the fourth membrane spanning segment (IVS4). Three CaCh 2 variants were cloned, subtypes 2a, 2c, and 2d, that are homologous to the a, c, and d variants of CaCh 3. For the skeletal muscle calcium channel only two variants were isolated. They are homologous to those of the a and c subtypes of CaCh 2 or 3, in that they differ only in the size of the IVS3 to IVS4 linker. These results demonstrate that calcium channel diversity is created by both the expression of distinct genes and the alternate splicing of these genes. 相似文献
6.
The complete amino acid sequence of a novel calcium channel (designated BII) from rabbit brain has been deduced by cloning and sequencing the cDNA. The BII calcium channel is structurally more closely related to the BI calcium channel than to the cardiac and skeletal muscle L-type calcium channels. Blot hybridization analysis of RNA from different tissues and from different regions of the brain shows that the BII calcium channel is distributed predominantly in the brain, being abundant in the cerebral cortex, hippocampus and corpus striatum. 相似文献
7.
Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel 总被引:20,自引:0,他引:20
The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopus oocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2(+)-dependent Cl- current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA. 相似文献
8.
Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord, and retina 总被引:9,自引:0,他引:9
Monoclonal antibodies that recognize the alpha 2 delta subunits of calcium channels from skeletal muscle immunoprecipitate a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. They also immunoprecipitate 64% of rabbit brain dihydropyridine-sensitive calcium channels followed by immunoprecipitation reveals alpha 1-, alpha 2 delta-, and beta-like subunits that have apparent molecular masses of 175, 142, and 57 kd, respectively. A polypeptide of 100 kd is also specifically immunoprecipitated. Immunocytochemical studies identify dihydropyridine-sensitive calcium channels in neuronal somata and proximal dendrites in rat brain, spinal cord, and retina. Staining of many neuronal somata is uneven, revealing relatively high densities of dihydropyridine-sensitive calcium channels at the base of major dendrites. L-type calcium channels in this location may serve to mediate long-lasting increases in intracellular calcium in the cell body in response to excitatory inputs to the dendrites. 相似文献
9.
Subunits of purified calcium channels. Alpha 2 and delta are encoded by the same gene 总被引:2,自引:0,他引:2
Polyacrylamide gel electrophoresis of purified rabbit skeletal muscle L-type calcium channel before and after reduction of disulfide bonds confirmed that 27- and 24-kDa forms of the delta subunit are disulfide-linked to the 143-kDa alpha 2 subunit. The amino acid sequences of three peptides obtained by tryptic digestion of the delta subunits corresponded to amino acid sequences predicted from the 3' region of the mRNA encoding alpha 2. One of these peptides had the same sequence as the N terminus of the 24- and 27-kDa forms of the delta subunit and corresponded to residues 935-946 of the predicted alpha 2 primary sequence. Anti-peptide antibodies directed to regions on the N-terminal side of this site recognized the 143-kDa alpha 2 subunit in immunoblots of purified calcium channels under reducing conditions, whereas an antipeptide antibody directed toward a sequence on the C-terminal side of this site recognized 24- and 27-kDa forms of the delta subunit. A similar result was obtained after immunoblotting using purified transverse tubules or crude microsomal membrane preparations indicating that alpha 2 and delta occur as distinct disulfide-linked polypeptides in skeletal muscle membranes. Thus, the delta subunits are encoded by the same gene as the alpha 2 subunit and are integral components of the skeletal muscle calcium channel. 相似文献
10.
Oussama El Far Béatrice Marquèze Christian Leveque Nicole Martin-Moutôt Bethan Lang John Newsom-Davis †Akira Yoshida †Masami Takahashi Michael J. Seagar 《Journal of neurochemistry》1995,64(4):1696-1702
Abstract: In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (125 I-ω conotoxin GVIA) and L-type ([3 H]PN200-110) calcium channels. Sera with a high antibody titer (>3 n M ) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin. 相似文献
11.
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. 相似文献
12.
Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers 总被引:19,自引:0,他引:19
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Vandebrouck C Martin D Colson-Van Schoor M Debaix H Gailly P 《The Journal of cell biology》2002,158(6):1089-1096
Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers. 相似文献
13.
Koszela-Piotrowska I Choma K Bednarczyk P Dołowy K Szewczyk A Kunz WS Malekova L Kominkova V Ondrias K 《Cellular & molecular biology letters》2007,12(4):493-508
Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane
potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study
was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane.
The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes
from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM
KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain).
The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives:
4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS).
The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain
mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited
by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive
to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells. 相似文献
14.
Deletion of amino acids 1641-2437 from the foot region of skeletal muscle ryanodine receptor alters the conduction properties of the Ca release channel. 总被引:2,自引:0,他引:2
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The ryanodine receptor (RyR) of skeletal muscle contains two functional domains: a carboxyl-terminal hydrophobic domain that forms the putative conduction pore of the calcium release channel, and a large cytoplasmic domain that corresponds to the "foot structure." To understand the contribution of the foot structure to the function of the calcium release channel, we studied a RyR deletion mutant, delta(1641-2437)-RyR, in which a region that is rich in glutamate and aspartate residues (a.a. 1641-2437) was removed. The wild-type and delta(1641-2437)-RyR proteins were expressed in a Chinese hamster ovary (CHO) cell line, and functions of single calcium release channels were measured in the lipid bilayer membrane. The wild-type RyR forms functional calcium release channels with a linear current-voltage relationship similar to that of the native channel identified in the sarcoplasmic reticulum membrane of skeletal muscle, whereas the channels formed by delta(1641-2437)-RyR exhibit significant inward rectification, i.e., currents moving from cytoplasm into SR lumen were approximately 20% less than that in the opposite direction. As in to the wt-RyR channel, opening of the delta(1641-2437)-RyR channel has a bell-shaped dependence on the cytoplasmic calcium, but the calcium-dependent activation and inactivation processes of the delta(1641-2437)-RyR channel are shifted to higher calcium concentrations. Our data show that deletion of a.a. 1641-2437 from the foot region of the skeletal muscle RyR results in changes in both ion conduction and calcium-dependent regulation of the calcium release channel. 相似文献
15.
P. V. Avdonin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2012,6(2):159-168
Modern data concerning expression, localization, biophysical properties, involvement in calcium regulation, and physiological
functions of TRP and Orai channels in skeletal muscle cells are analyzed. In skeletal muscles TRPC1/2/3/4/5/6/7, TRPV2/4,
TRPM2/7 and Orai1/3 channels are expressed. Activities of TRPC1/3 and TRPV4 facilitate maximal muscle contraction during tetanus.
Orai1 channels provide recovery of intracellular calcium stores and are obligatory for proliferation of myoblasts and differentiation
of skeletal muscles. TRPC1 knockout results in alterations of the development of skeletal muscles. Enhanced calcium influx
via the channels is supposed to be a pathogenic factor of myodystrophy. 相似文献
16.
R J Murphy L Béliveau P F Gardiner A Calderone 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1999,221(3):184-187
The mechanism(s) responsible for beta2-adrenergic receptor-mediated skeletal muscle and cardiac hypertrophy remains undefined. This study examined whether calcium influx through L-type calcium channels contributed to the development of cardiac and skeletal muscle (plantaris; gastrocnemius; soleus) hypertrophy during an 8-day treatment with the beta2-adrenergic receptor agonist clenbuterol. Concurrent blockade of L-type calcium channels with nifedipine did not reverse the hypertrophic action of clenbuterol. Moreover, nifedipine treatment alone resulted in both cardiac and soleus muscle hypertrophy (6% and 7%, respectively), and this effect was additive to the clenbuterol-mediated hypertrophy in the heart and soleus muscles. The hypertrophic effects of nifedipine were not associated with increases in total beta-adrenergic receptor density, nor did nifedipine reverse clenbuterol-mediated beta-adrenergic receptor downregulation in either the left ventricle or soleus muscle. Both nifedipine and clenbuterol-induced hypertrophy increased total protein content of the soleus and left ventricle, with no change in protein concentration. In conclusion, our results support the hypothesis that beta2-adrenergic receptor agonist-induced muscle hypertrophy is mediated by mechanisms other than calcium influx through L-type calcium channels. 相似文献
17.
A cDNA clone encoding a protein with high homology to the beta-subunit of the rabbit skeletal muscle dihydropyridine-sensitive calcium channel was isolated from a rat brain cDNA library. This rat brain beta-subunit cDNA hybridizes to a 3.4 kb message that is expressed in high levels in the cerebral hemispheres and hippocampus but is significantly reduced in cerebellum. The open reading frame encodes 597 amino acids with a predicted mass of 65 679 Da which is 82% homologous with the skeletal muscle beta-subunit. The brain cDNA encodes a unique 153 amino acid C-terminus and predicts the absence of a muscle-specific 50 amino acid internal segment. It also encodes numerous consensus phosphorylation sites suggesting a role in calcium channel regulation. The corresponding human beta-subunit gene was localized to chromosome 17. Hence the encoded brain beta-subunit, which has a primary structure highly similar to its isoform in skeletal muscle, may have a comparable role as an integral regulatory component of a neuronal calcium channel. 相似文献
18.
P D Syme F Brunotte Y Green J K Aronson G K Radda 《Biochimica et biophysica acta》1991,1093(2-3):234-240
We have studied the in vivo response of the Na+/H+ antiporter in skeletal muscle to beta 2-adrenoceptor stimulation with isoprenaline and the effect of blocking L-type calcium channels with nifedipine. Na+/H+ antiporter activity in skeletal muscle in vivo increased after beta 2-adrenoceptor stimulation with isoprenaline; nifedipine attenuated that effect. This suggests that opening of L-type calcium channels is necessary for full activation of the Na+/H+ antiporter in skeletal muscle. Bleeding also increased Na/H+ antiporter activity, which we believe could be explained by an increase in sympathetic nervous system activity as a result of hypotension. This may be one of the mechanisms by which animals under stress prepare their skeletal muscle for exercise as part of the 'fright and flight' reaction. 相似文献
19.
The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding. 相似文献
20.
A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel. 相似文献