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1.
The skeletal muscle (SKM) L-type Ca2+ channel is composed of a central subunit designated alpha 1, which contains the pore and the dihydropyridine (DHP) binding domains and three associated subunits, alpha 2/delta, beta, and gamma, which influence the activity of the SKM alpha 1. Coexpression of SKM alpha 1 and SKM beta in stably transfected mouse L cells results in a dramatic increase in DHP binding accompanied by fast gated Ba2+ currents. We report here that this "SKM alpha 1 beta-related phenotype" can be converted upon intracellular trypsin treatment into a slowly inactivating, DHP sensitive "SKM alpha 1 phenotype." These observations indicate that current amplitude, fast inactivation, and DHP sensitivity are modulated by an interaction of SKM alpha 1 and SKM beta on the internal side of the membrane. 相似文献
2.
D Rampe H S Kim A E Lacerda L Birnbaumer A M Brown 《Biochemical and biophysical research communications》1990,169(3):825-831
We examined the binding of the 1,4-dihydropyridine (DHP) [3H]PN200-110 to membranes from a fibroblast cell line transfected with the alpha 1 subunit (DHP receptor) of the L-type Ca2+ channel from rabbit skeletal muscle. Binding site affinity (KD) and density (Bmax) were 1.16 +/- 0.31 nM and 142 +/- 17 fmoles/mg protein, respectively. This affinity corresponded closely with that observed in native skeletal muscle. The Ca2+ channel antagonists diltiazem and MDL 12,330A stimulated [3H]PN200-110 binding in a dose-dependent manner while flunarizine, quinacrine and trifluoperazine inhibited binding. Surprisingly, D600 also stimulated [3H]PN200-110 binding in a dose-dependent and stereoselective manner. It is concluded that the fibroblast cells used in this study provide a unique system for interactions of the Ca2+ channel ligands with the alpha 1 subunit of the skeletal muscle L-type Ca2+ channel. 相似文献
3.
Yamaguchi S Okamura Y Nagao T Adachi-Akahane S 《The Journal of biological chemistry》2000,275(52):41504-41511
The dihydropyridine (DHP)-binding site has been identified within L-type Ca(2+) channel alpha(1C) subunit. However, the molecular mechanism underlying modulation of Ca(2+) channel gating by DHPs has not been clarified. To search for novel determinants of high affinity DHP binding, we introduced point mutations in the rat brain Ca(2+) channel alpha(1C) subunit (rbCII or Ca(v)1.2c) based on the comparison of amino acid sequences between rbCII and the ascidian L-type Ca(2+) channel alpha(1) subunit, which is insensitive to DHPs. The alpha(1C) mutants (S1115A, S1146A, and A1420S) and rbCII were transiently expressed in BHK6 cells with beta(1a) and alpha(2)/delta subunits. The mutation did not affect the electrophysiological properties of the Ca(2+) channel, or the voltage- and concentration-dependent block of Ca(2+) channel currents produced by diltiazem and verapamil. However, the S1115A channel was significantly less sensitive to DHP antagonists. Interestingly, in the S1115A channel, DHP agonists failed to enhance whole-cell Ca(2+) channel currents and the prolongation of mean open time, as well as the increment of NP(o). Responsiveness to the non-DHP agonist FPL-64176 was also markedly reduced in the S1115A channel. When S1115 was replaced by other amino acids (S1115D, S1115T, or S1115V), only S1115T was slightly sensitive to S-(-)-Bay K 8644. These results indicate that the hydroxyl group of Ser(1115) in IIIS5-S6 linker of the L-type Ca(2+) channel alpha(1C) subunit plays a critical role in DHP binding and in the action of DHP Ca(2+) channel agonists. 相似文献
4.
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
5.
Calcium channel blockers bind with high affinity to sites on the voltage-sensitive Ca2+ channel. Radioligand binding studies with various Ca2+ channel blockers have facilitated identification and characterization of binding sites on the channel structure. In the present study we evaluated the relationship between the binding sites for the Ca2+ channel blockers on the voltage-sensitive Ca2+ channel from rabbit heart sarcolemma and rabbit skeletal muscle transverse tubules. [3H]PN200-110 binds with high affinity to a single population of sites on the voltage-sensitive Ca2+ channel in both rabbit heart sarcolemma and skeletal muscle transverse tubules. [3H]PN200-110 binding was not affected by added Ca2+ whereas EGTA and EDTA noncompetitively inhibited binding in both types of membrane preparations. EDTA was a more potent inhibitor of [3H]PN200-110 binding than EGTA. Diltiazem stimulates the binding of [3H]PN200-110 in a temperature-sensitive manner. Verapamil inhibited binding of [3H]PN200-110 to both types of membrane preparations in a negative manner, although this effect was of a complex nature in skeletal muscle transverse tubules. The negative effect of verapamil on [3H]PN200-110 binding in cardiac muscle was completely reversed by Ca2+. On the other hand, Ca2+ was without effect on the negative cooperativity seen between verapamil and [3H]PN200-110 binding in skeletal muscle transverse tubules. Since Ca2+ did not affect [3H]PN200-110 binding to membranes, we would like to suggest that Ca2+ is modulating the negative effect of verapamil on [3H]PN200-110 binding through a distinct Ca2+ binding site.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines. 相似文献
7.
Interaction of charybdotoxin S10A with single maxi-K channels: kinetics of blockade depend on the presence of the beta 1 subunit 总被引:1,自引:0,他引:1
The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, ChTX-S10A block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction. 相似文献
8.
Modulation of the Ca2+ channel voltage sensor and excitation-contraction coupling by silver.
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Ag+ (0.5-10 microM) is known to produce a transient contraction of intact frog skeletal muscle fibers followed by complete inhibition of excitation-contraction (E-C) coupling. We have carried out physiological and biochemical experiments to investigate the basis of this effect. Dihydropyridine (DHP) Ca2+ channel blockers, which inhibit the voltage sensor of the Ca2+ channel, completely inhibit Ag+ contractions. Removal of extracellular Ca2+, or blockade of Ca2+ entry with cadmium, does not inhibit Ag+ contractions. Activation of the Ca2+ channel's voltage sensor with the Ca2+ channel agonists Bay K 8644 or with perchlorate, potentiates the Ag(+)-induced contraction. Ag+ binds to the partially purified rabbit skeletal muscle Ca2+ channel and inhibits DHP binding (IC50 = 1.1 microM) and sulfhydryl (SH) reactivity (IC50 = 0.11 microM) over the concentration range where it inhibits E-C coupling. Oxidation of free SH groups by H2O2 or their reaction with DTNB prevents Ag+ contractions, while DTT reduction of oxidized SH groups restores Ag+ contractions. These results suggest that Ag+ binds to critical SH groups on the DHP receptor Ca2+ channel, resulting in modification of the channel's voltage sensor and the failure of E-C coupling. 相似文献
9.
E Perez-Reyes A Castellano H S Kim P Bertrand E Baggstrom A E Lacerda X Y Wei L Birnbaumer 《The Journal of biological chemistry》1992,267(3):1792-1797
The skeletal muscle dihydropyridine receptor/Ca2+ channel is composed of five protein components (alpha 1, alpha 2 delta, beta, and gamma). Only two such components, alpha 1 and alpha 2, have been identified in heart. The present study reports the cloning and expression of a novel beta gene that is expressed in heart, lung, and brain. Coexpression of this beta with a cardiac alpha 1 in Xenopus oocytes causes the following changes in Ca2+ channel activity: it increases peak currents, accelerates activation kinetics, and shifts the current-voltage relationship toward more hyperpolarized potentials. It also increases dihydropyridine binding to alpha 1 in COS cells. These results indicate that the cardiac L-type Ca2+ channel has a similar subunit structure as in skeletal muscle, and provides evidence for the modulatory role of the beta subunit. 相似文献
10.
S L Hamilton J Codina M J Hawkes A Yatani T Sawada F M Strickland S C Froehner A M Spiegel L Toro E Stefani 《The Journal of biological chemistry》1991,266(29):19528-19535
The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs. 相似文献
11.
Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release 总被引:10,自引:0,他引:10
Catterall WA 《Cell calcium》1998,24(5-6):307-323
Electrophysiological studies of neurons reveal different Ca2+ currents designated L-, N-, P-, Q-, R-, and T-type. High-voltage-activated neuronal Ca2+ channels are complexes of a pore-forming alpha 1 subunit of about 190-250 kDa, a transmembrane, disulfide-linked complex of alpha 2 and delta subunits, and an intracellular beta subunit, similar to the alpha 1, alpha 2 delta, and beta subunits previously described for skeletal muscle Ca2+ channels. The primary structures of these subunits have all been determined by homology cDNA cloning using the corresponding subunits of skeletal muscle Ca2+ channels as probes. In most neurons, L-type channels contain alpha 1C or alpha 1D subunits, N-type contain alpha 1B subunits, P- and Q-types contain alternatively spliced forms of alpha 1A subunits, R-type contain alpha 1E subunits, and T-type contain alpha 1G or alpha 1H subunits. Association with different beta subunits also influences Ca2+ channel gating substantially, yielding a remarkable diversity of functionally distinct molecular species of Ca2+ channels in neurons. 相似文献
12.
Dihydropyridine and phenylalkylamine receptors associated with cardiac and skeletal muscle calcium channels are structurally different 总被引:1,自引:0,他引:1
We have purified putative L-type Ca2+ channels from chick heart by virtue of their associated high affinity receptors for the Ca2+ channel effectors, dihydropyridines (DHPs), and phenylalkylamines (PAAs). A peptide of 185,000-190,000 daltons was found to comigrate with the peak of DHP binding activity during purification through two successive cycles of lectin affinity chromatography and sucrose density gradient centrifugation. A previously described peptide of 140,000 daltons, whose Mr was increased to approximately 180,000 under nonreducing conditions, also copurified with the 185-kDa peptide and dihydropyridine binding activity. When cardiac membranes were photolabeled with either the dihydropyridine [3H]azidopine or the PAA [3H]azidopamil prior to purification, a single, specifically labeled component of 185,000-190,000 daltons was present in the purified fractions. The properties of this 185-kDa cardiac DHP/PAA receptor were compared to the smaller 165-kDa DHP/PAA receptor previously purified from skeletal muscle. Antibodies raised against the 165-kDa skeletal muscle DHP/PAA receptor reacted with both rabbit and chick skeletal muscle receptors, but only poorly recognized, if at all, the cardiac 185-190 kDa component. The 185-kDa peptide present in the purified fractions obtained from cardiac muscle did not undergo substantial phosphorylation by cAMP-dependent protein kinase, while the purified 165-kDa peptide from rabbit and chick skeletal muscle was a good substrate for this kinase. The results show that the DHP and PAA receptors in cardiac muscle are contained in a 185-190-kDa peptide that is significantly larger than, and structurally and immunologically different from, it skeletal muscle counterpart. 相似文献
13.
Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor beta 1 subunit. 总被引:1,自引:0,他引:1
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The Ca2+ currents, charge movements, and intracellular Ca2+ transients in mouse skeletal muscle cells homozygous for a null mutation in the cchb1 gene encoding the beta 1 subunit of the dihydropyridine receptor have been characterized. I beta null, the L-type Ca2+ current of mutant cells, had a approximately 13-fold lower density than the L-type current of normal cells (0.41 +/- 0.042 pA/pF at + 20 mV, compared with 5.2 +/- 0.38 pA/pF in normal cells). I beta null was sensitive to dihydropyridines and had faster kinetics of activation and slower kinetics of inactivation than the L-type current of normal cells. Charge movement was reduced approximately 2.8-fold, with Qmax = 6.9 +/- 0.61 and Qmax = 2.5 +/- 0.2 nC/microF in normal and mutant cells, respectively. Approximately 40% of Qmax was nifedipine sensitive in both groups. In contrast to normal cells, Ca2+ transients could not be detected in mutant cells at any test potential; however, caffeine induced a robust Ca2+ transient. In homogenates of mutant muscle, the maximum density of [3H]PN200-110 binding sites (Bmax) was reduced approximately 3.9-fold. The results suggest that the excitation-contraction uncoupling of beta 1-null skeletal muscle involves a failure of the transduction mechanism that is due to either a reduced amount of alpha 1S subunits in the membrane or the specific absence of beta 1 from the voltage-sensor complex. 相似文献
14.
Subunit composition of the purified dihydropyridine binding protein from skeletal muscle 总被引:1,自引:0,他引:1
The dihydropyridine (DHP) receptor from rabbit skeletal muscle has been characterized by affinity labeling and purification. Two procedures were used for purification: one that was a procedure modified from that of Curtis and Catterall (1984) and one that employed an anti alpha 1 monoclonal antibody (Mab) affinity column. In addition, both digitonin and CHAPS solubilizations were utilized with each purification technique. The major findings are as follows: (1) In contrast to the behavior in digitonin, neither the 52K (beta) nor the 140K (alpha 2) polypeptide quantitatively copurifies with the 170K (alpha 1) polypeptide when the purification is carried out in CHAPS. This has been shown by use of both wheat germ and monoclonal antibody columns. The digitonin-extracted receptor complex bound to the Mab affinity column loses alpha 2 and beta when the digitonin is replaced by CHAPS, and when the complex is bound to a WGA column, a CHAPS wash causes dissociation of alpha 1, beta, and gamma from alpha 2. Loss of binding of dihydropyridines occurs with the CHAPS wash but can be partially restored by the addition of the CHAPS wash to the material eluted from the column with N-acetylglucosamine. (2) Although both detergents solubilized greater than 80% of the polypeptides associated with the DHP binding site, the ability of these proteins to bind dihydropyridines is reduced more by CHAPS treatment than by digitonin treatment, raising the possibility that subunit interactions contribute to high-affinity binding. Alternatively, CHAPS may remove tightly bound lipids necessary for binding or cause irreversible denaturation of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
Regulation of skeletal muscle dihydropyridine receptor gene expression by biomechanical unloading. 总被引:6,自引:0,他引:6
S Kandarian S O'Brien K Thomas L Schulte J Navarro 《Journal of applied physiology》1992,72(6):2510-2514
Biomechanical unloading of the rat soleus by hindlimb unweighting is known to induce atrophy and a slow- to fast-twitch transition of skeletal muscle contractile properties, particularly in slow-twitch muscles such as the soleus. The purpose of this study was to determine whether the expression of the dihydropyridine (DHP) receptor gene is upregulated in unloaded slow-twitch soleus muscles. A rat DHP receptor cDNA was isolated by screening a random-primed cDNA lambda gt10 library from denervated rat skeletal muscle with oligonucleotide probes complementary to the coding region of the rabbit DHP receptor cDNA. Muscle mass and DHP receptor mRNA expression were assessed 1, 4, 7, 14, and 28 days after hindlimb unweighting in rats by tail suspension. Isometric twitch contraction times of soleus muscles were measured at 28 days of unweighting. Northern blot analysis showed that tissue distribution of DHP receptor mRNA was specific for skeletal muscle and expression was 200% greater in control fast-twitch extensor digitorum longus (EDL) than in control soleus muscles. A significant stimulation (80%) in receptor message of the soleus was induced as early as 24 h of unloading without changes in muscle mass. Unloading for 28 days induced marked atrophy (control = 133 +/- 3 vs. unweighted = 62.4 +/- 1.8 mg), and expression of the DHP receptor mRNA in the soleus was indistinguishable from levels normally expressed in EDL muscles. These changes in mRNA expression are in the same direction as the 37% reduction in time to peak tension and 28% decrease in half-relaxation time 28 days after unweighting. Our results suggest that muscle loading necessary for weight support modulates the expression of the DHP receptor gene in the soleus muscle. 相似文献
16.
17.
Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca2+ release channel. Three lipids, N,N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [3H]ryanodine binding by greatly increasing the rate of dissociation of bound [3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [3H]ryanodine binding. These bases also decreased the activity of the Ca2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO4 and C6 to C18 ceramides of sphingosine had no significant effect on [3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4-5 decreased the ability of the sphingolipid bases to inhibit [3H]ryanodine binding 2-3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca2+ release channel and as a class possess a common mechanism of inhibition. 相似文献
18.
Two domains in dihydropyridine receptor activate the skeletal muscle Ca(2+) release channel
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The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs. 相似文献
19.
Differential regulation of skeletal muscle L-type Ca2+ current and excitation-contraction coupling by the dihydropyridine receptor beta subunit
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Beurg M Sukhareva M Ahern CA Conklin MW Perez-Reyes E Powers PA Gregg RG Coronado R 《Biophysical journal》1999,76(4):1744-1756
The dihydropyridine receptor (DHPR) of skeletal muscle functions as a Ca2+ channel and is required for excitation-contraction (EC) coupling. Here we show that the DHPR beta subunit is involved in the regulation of these two functions. Experiments were performed in skeletal mouse myotubes selectively lacking a functional DHPR beta subunit. These beta-null cells have a low-density L-type current, a low density of charge movements, and lack EC coupling. Transfection of beta-null cells with cDNAs encoding for either the homologous beta1a subunit or the cardiac- and brain-specific beta2a subunit fully restored the L-type Ca2+ current (161 +/- 17 pS/pF and 139 +/- 9 pS/pF, respectively, in 10 mM Ca2+). We compared the Boltzmann parameters of the Ca2+ conductance restored by beta1a and beta2a, the kinetics of activation of the Ca2+ current, and the single channel parameters estimated by ensemble variance analysis and found them to be indistinguishable. In contrast, the maximum density of charge movements in cells expressing beta2a was significantly lower than in cells expressing beta1a (2.7 +/- 0.2 nC/microF and 6.7 +/- 0. 4 nC/microF, respectively). Furthermore, the amplitude of Ca2+ transient measured by confocal line-scans of fluo-3 fluorescence in voltage-clamped cells were 3- to 5-fold lower in myotubes expressing beta2a. In summary, DHPR complexes that included beta2a or beta1a restored L-type Ca2+ channels. However, a DHPR complex with beta1a was required for complete restoration of charge movements and skeletal-type EC coupling. These results suggest that the beta1a subunit participates in key regulatory events required for the EC coupling function of the DHPR. 相似文献
20.
The dihydropyridine receptor is associated with the L-type Ca2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC50 value of 50 microM, while DTT inhibited maximally by 75% with an IC50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [3H]PN200-110 binding in cardiac sarcolemma approx. 40-60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC50 value of 20 microM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca2+/calmodulin-dependent phosphorylation increased [3H]nitrendipine binding 20% with no alteration in KD in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca2+ channels. 相似文献