Characteristics of specific bindings of nitrendipine and PN200-110 to various crude membranes: induction of irreversible bindings by UV irradiation

The characteristics of the specific bindings of [3H]nitrendipine (Nit) and [3H](+)PN200-110 (PN) to crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain were investigated, with special interest in the effect of UV irradiation on these bindings. The specific bindings of [3H]Nit and [3H](+)PN to these crude membranes were saturable and reversible. The specific bindings of [3H]Nit to all these membranes except crude skeletal membranes was maximum in the presence of 0.15 M NaCl plus 1 mM CaCl2 and minimal in the absence of these ions, but the specific bindings of [3H](+)PN to these crude membranes was not affected significantly by these ions. A calcium agonist and antagonists inhibited the specific bindings of [3H]Nit and [3H](+)PN to these crude membranes, the order of their inhibitory effects on specific [3H]Nit bindings being roughly Nit greater than or equal to (+)PN greater than or equal to (-)PN much greater than Bay K 8644 (Bay) greater than verapamil (Ver) greater than diltiazem (Dil). In crude skeletal membranes only, PN caused significant stereospecific inhibition. The order of inhibitions of specific [3H](+)PN bindings to these crude membranes was generally (+)PN greater than Nit greater than or equal to (-)PN greater than Bay much greater than Ver greater than or equal to Dil. In all these crude membranes, UV irradiation completely prevented decrease in the amount of specific binding of [3H](+)PN binding on addition of excess unlabeled (+)PN. These findings suggested that [3H]Nit and [3H](+)PN bind to voltage-sensitive calcium channels in crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain, and that UV irradiation changes the specific bindings of [3H]Nit and [3H](+)PN from reversible to irreversible bindings.     

Effects of GTP analogues and activation of endogenous protein kinases on photoaffinity labeling with [3H](+)PN200-110 of crude membranes from rat heart and brain.

The effects of GTP analogues and conditions in which various endogenous protein kinases were activated on photoaffinity labeling with [3H](+)PN200-110 (PN) of crude membranes from rat cardiac muscle and whole brain were investigated. Photoaffinity labeling with 20 nM [3H](+)PN of these crude membranes was decreased by 100 microM GTP-gamma-S, but not by 100 microM GTP or 100 microM GDP-beta-S. Similar results were obtained on the effects of GTP and its analogues on the specific binding of 20 nM [3H](+)PN to these crude membranes under the same conditions. Activation of endogenous protein kinases in these crude membranes did not influence the photoaffinity labeling with [3H](+)PN. These results suggested the binding sites, or DPH-sensitive, or L-type, calcium channels in curde membranes from rat cardiac muscle and whole brain are directly or indirectly modulated by endogenous GTP-binding protein, but not by various endogenous protein kinases in these crude membranes.     

Identification of the benzothiazepine-binding polypeptide of skeletal muscle calcium channels with (+)-cis-azidodiltiazem and anti-ligand antibodies

The purified dihydropyridine-sensitive calcium channel from skeletal muscle transverse tubules consists of several subunits, termed alpha 1, alpha 2, beta, gamma and delta. From its associated drug receptors, those for 1,4-dihydropyridines and phenylalkylamines have been shown previously by photoaffinity labeling to reside on the alpha 1 subunit. In the present study the arylazide photo-affinity ligand, (+)-cis-azidodiltiazem ((+)-cis-(2S,3S)-5-[2-(4- azidobenzoyl)aminoethyl]-2,3,4,5-tetrahydro-3-hydroxy-2-(4-methoxyphenyl )-4- oxo-1,5-benzothiazepine), and the respective tritiated derivative, (+)-cis-[3H]azidodiltiazem (45 Ci/mmol), were developed to identify directly the benzothiazepine binding subunit. (+)-cis-Azidodiltiazem binds competitively to the benzothiazepine receptor in rabbit skeletal muscle transverse tubule membranes. Upon ultraviolet irradiation of the (+)-cis-[3H]azidodiltiazem-purified calcium channel complex, the ligand photoincorporates exclusively into the alpha 1 subunit. Photoincorporation is protected by 100 microM (-)-desmethoxyverapamil and 100 microM (+)-cis-diltiazem. A polyclonal antiserum directed against (+)-cis-azidodiltiazem was employed to detect (+)-cis-azidodiltiazem immunoreactivity photoincorporated into the purified calcium channel complex, confirming the exclusive labeling of the alpha 1 subunit. Our data provide direct evidence that, together with the drug receptors for 1,4-dihydropyridines and phenylalkylamines, the benzothiazepine binding domain of skeletal muscle calcium channels is located on the alpha 1 subunit. We conclude that our anti-ligand antibodies could be used successfully to affinity purify the photolabeled proteolytic fragments of the alpha 1 subunit which are expected to form part of the benzothiazepine binding domain.     

Specific bindings of [3H](+)PN200-110 and [125I]ω-conotoxin to crude membranes from differentiated NG108-15 cells

The characteristics of the specific bindings of [³H](+)PN200-110 (PN: L-type Ca channel antagonist) and [¹²⁵I]-conotoxin G VI A (-CgTX: neuronal L-or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma x glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt₂cAMP) were examined, because we have already observed that the magnitude and rate of KCL-stimulated⁴⁵Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt₂cAMP (unpublished results). The specific binding of [³H](+)PN to these crude membranes was saturable at various concentrations of 2.5–5.0 nM [³H](+)PN. Scatchard analysis showed that the specific binding of [³H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt₂cAMP, but that the apparent Kd value for the specific binding of [³H](+)PN was not influenced by treatment with Bt₂cAMP. The specific binding of [³H](+)PN to crude membranes from Bt₂cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN>nitrendipine>(–)PNBay K 8644diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [³H(+)PN. On the other hand, [¹²⁵I]-CgTX at concentrations of 0.075–0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions. These results suggest that the increase in magnitude or rate of KCl-stimulated⁴⁵Ca uptake during differentiation of NG108-15 cells is partially due to quantitative alteration of voltage-sensitive Ca channels in the cells, and that there are scarcely any specific binding sites for [¹²⁵I]-CgTX on Bt₂cAMP-treated or untreated NG108-15 cells.     

Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol

Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).     

Specific derivatization of the vesicle monoamine transporter with novel carrier-free radioiodinated reserpine and tetrabenazine photoaffinity labels

Two iodophenylazide derivatives of reserpine and one iodophenylazide derivative of tetrabenazine have been synthesized and characterized as photoaffinity labels of the vesicle monoamine transporter (VMAT2). These compounds are 18-O-[3-(3'-iodo-4'-azidophenyl)-propionyl]methyl reserpate (AIPPMER), 18-O-[N-(3'-iodo-4'-azidophenethyl)glycyl]methyl reserpate (IAPEGlyMER), and 2-N-[(3'-iodo-4'-azidophenyl)-propionyl]tetrabenazine (TBZ-AIPP). Inhibition of [3H]dopamine uptake into purified chromaffin granule ghosts showed IC50 values of approximately 37 nM for reserpine, 83 nM for AIPPMER, 200 nM for IAPEGlyMER, and 2.1 microM for TBZ-AIPP. Carrier-free radioiodinated [125I]IAPEGlyMER and [125I]TBZ-AIPP were synthesized and used to photoaffinity label chromaffin granule membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed specific [125I]IAPEGlyMER labeling of a polypeptide that migrated as a broad band (approximately 55-90 kDa), with the majority of the label located between 70 and 80 kDa. The labeling by [125I]IAPEGlyMER was blocked by 100 nM reserpine, 10 microM tetrabenazine, 1 mM serotonin, and 10 mM (-)-norepinephrine and dopamine. Analysis of [125I]TBZ-AIPP-labeled chromaffin granule membranes by SDS-PAGE and autoradiography demonstrated specific labeling of a similar polypeptide, which was blocked by 1 microM reserpine and 10 microM tetrabenazine. Incubation of [125I]TBZ-AIPP-photolabeled chromaffin granule membranes in the presence of the glycosidase N-glycanase shifted the apparent molecular weight of VMAT2 to approximately 51 kDa. These data indicate that [125I]IAPEGlyMER and [125I]TBZ-AIPP are effective photoaffinity labels for VMAT2.     

Photoaffinity labeling of the calcium channel antagonist receptor in the heart of the cardiomyopathic hamster

The high affinity 1,4-dihydropyridine receptors of the cardiac membrane calcium channel from Syrian Cardiomyopathic hamsters were studied using [3H] PN200-110 and [3H]azidopine as ligands. [3H]Azidopine was photoincorporated covalently into bands of 180, 100, 79, 45 and 31 kDa, as determined by SDS/polyacrylamide gel electrophoresis. Photolabeling of the 180 kDa band is protected by 2 microM [1H]PN200-110 whereas the lower Mr bands are not. Thus, only the 180 kDa band is the calcium channel linked 1,4 dihydropyridine receptor. The photoincorporation into this 180 kDa band is doubled with samples of myopathic hamsters vs. control hamsters. It is suggested that the increase in calcium channel receptors may be involved in the pathogenesis of this cardiomyopathy.     

Photoaffinity labeling of A1-adenosine receptors

The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for [3H]N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [3H]N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity (125I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for 125I-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that 125I-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.     

(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.     

Comparative Effects of Chronic Exposure to Ethanol and Calcium Channel Antagonists on Calcium Channel Antagonist Receptors in Cultured Neural (PC12) Cells

Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)-[3H]PN 200-110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long-term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)-[3H]PN 200-110 binding to PC12 membranes. Cotreatment of ethanol-containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)-[3H]PN 200-110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.     

Stereoselective photoaffinity labelling of the purified 1,4-dihydropyridine receptor of the voltage-dependent calcium channel

The voltage-dependent calcium channel from guinea-pig skeletal muscle T-tubules has been isolated with a rapid, two-step purification procedure. Reversible postlabelling of the channel-linked 1,4-dihydropyridine receptor and stereoselective photolabelling as a novel approach were employed to assess purity. A 135-fold purification to a specific activity of 1311 +/- 194 pmol/mg protein (determined by reversible equilibrium binding with (+)-[3H]PN200-110) was achieved. Three polypeptides of 155 kDa, 65 kDa and 32 kDa were identified in the purified preparation. The 155-kDa band is a glycoprotein. The arylazide photoaffinity probe (-)-[3H]azidopine bound with high affinity to solubilized membranes (Kd = 0.7 +/- 0.2 nM) and highly purified fractions (Kd = 3.1 +/- 2 nM), whereas the optical antipode (+)-azidopine was of much lower affinity. Irradiation of (-)-[3H]azidopine and (+)-[3H]azidopine receptor complexes with ultraviolet light led to preferential incorporation of the (-) enantiomer into the 155-kDa polypeptide in crude solubilized and purified preparations. The pharmacological profile of irreversible labelling of the 155-kDa glycoprotein by (-)-[3H]azidopine is identical to that found in reversible binding experiments. Specific photolabelling of the 155-kDa band by (-)-[3H]azidopine per milligram of protein increases 150-fold upon purification, whereas incorporation into non-specific bands in the crude solubilized material is identical for both, (-) and (+)-[3H]azidopine.     

Direct photoaffinity labeling of the high affinity nitrendipine-binding site in subcellular membrane fractions isolated from canine myocardium

[3H]Nitrendipine and high intensity ultraviolet irradiation have been used to photoaffinity label the protein component of the high affinity nitrendipine-binding site in subcellular membrane fractions from canine cardiac muscle. Irradiation of isolated cardiac membranes in the presence of [3H]nitrendipine resulted in the covalent labeling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 32,000. Incorporation of [3H]nitrendipine did not occur in the absence of irradiation. The photoaffinity labeling of the 32,000-Da protein by [3H]nitrendipine was inhibited by excess unlabeled nitrendipine, nifedipine, or verapamil. EDTA, ATP, and La3+, which are known to reduce high affinity nitrendipine binding, also inhibited the photoaffinity labeling of this membrane protein by [3H]nitrendipine. The 32,000-Da [3H]nitrendipine-labeled protein was found to be enriched in the ryanodine-sensitive fraction of cardiac sarcoplasmic reticulum and absent from the ryanodine-insensitive fraction of cardiac sarcoplasmic reticulum which is known to lack high affinity nitrendipine binding. Therefore, the 32,000-Da photoaffinity-labeled [3H]nitrendipine-binding protein exhibits properties identical to those expected for the protein component of the high affinity nitrendipine-binding site in isolated cardiac membranes.     

Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

Purified rat liver glucocorticoid receptor was covalently charged with [3H]glucocorticoid by photoaffinity labeling (UV irradiation of [3H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [3H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [3H]triamcinolone acetonide and Cys-656 after affinity labeling with [3H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.     

Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin

Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.     

Low-affinity binding sites for 1,4-dihydropyridines in skeletal muscle transverse tubule membranes revealed by changes in the fluorescence of felodipine.

The fluorescence changes accompanying the binding of the fluorescent calcium channel antagonist, felodipine, to transverse tubule membranes from rabbit skeletal muscle have been used to characterize low-affinity binding sites for 1,4-dihydropyridine derivatives in these preparations. In competition experiments, felodipine inhibited the high-affinity binding of (+)-[3H]PN200-110 to transverse tubule membranes with an apparent Ki of 5 +/- 2 nM. Binding of felodipine to additional low-affinity sites resulted in a large, saturable (Kd = 6 +/- 2 microM) increase in its fluorescence which could be excited either directly (380 nm) or indirectly via energy transfer from membrane protein (290 nm). The observed fluorescence enhancement was competitively inhibited by other 1,4-dihydropyridines with inhibition constants of 3-21 microM but was unaffected by the structurally unrelated calcium channel antagonists, diltiazem and verapamil, or by Ca2+, Cd2+, and La3+. Both high- and low-affinity binding sites appear to be localized in the transverse tubular system, since the magnitude of the observed fluorescence enhancement was higher in these membranes than in microsomal preparations and was directly proportional to the density of high-affinity sites for (+)-[3H]PN200-110. Furthermore, both high- and low-affinity sites appear to be conformationally coupled since, over the same concentration range that the fluorescence changes were observed, felodipine accelerated the rate of dissociation of [3H]PN200-110 previously bound to its high-affinity sites. Similar behavior has previously been reported for other 1,4-dihydropyridines [Dunn, S. M. J., & Bladen, C. (1991) Biochemistry 30, 5716-5721].(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple calcium channels in synaptosomes: voltage dependence of 1,4-dihydropyridine binding and effects on function

The voltage dependence of binding of the calcium channel antagonist, (+)-[3H]PN200-110, to rat brain synaptosomes and the effects of dihydropyridines on 45Ca2+ uptake have been investigated. Under nondepolarizing conditions (+)-[3H]PN200-110 binds to a single class of sites with a Kd of 0.07 nM and a binding capacity of 182 fmol/mg of protein. When the synaptosomal membrane potential was dissipated either by osmotic lysis of the synaptosomes or by depolarization induced by raising the external K+ concentration, there was a decrease in affinity (approximately 7-fold) with no change in the number of sites. The effects of calcium channel ligands on 45Ca2+ uptake by synaptosomes have been measured as a function of external potassium concentration, i.e., membrane potential. Depolarization led to a rapid influx of 45Ca2+ whose magnitude was voltage-dependent. Verapamil (100 microM) almost completely inhibited calcium uptake at all potassium concentrations studied. In contrast, the effects of dihydropyridines (2 microM) appear to be voltage-sensitive. At relatively low levels of depolarization (10-25 mM K+) nitrendipine and PN200-110 completely inhibited 45Ca2+ influx, whereas the agonist Bay K8644 slightly potentiated the response. At higher K+ concentrations an additional dihydropyridine-insensitive component of calcium uptake was observed. These results provide evidence for the presence of dihydropyridine-sensitive calcium channels in synaptosomes which may be activated under conditions of partial depolarization.     

Photoaffinity labelling of Ca2+ channels with [3H]azidopine

A 1,4-dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a Kd of 350 pM to guinea-pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited steroselectively by 1,4-dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of Mr approximately 145 000. The covalent labelling of the Mr approximately 145 000 band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.     

Photoaffinity labeling of serotonin-binding proteins

A photosensitive arylazide derivative of serotonin (nitroaryl-azidophenyl serotonin, NAP-serotonin) has been synthesized for use in studying the biochemical nature of serotonin binding sites. [³H]-NAP-serotonin possesses a similar ability to bind to the crude membranes of rat brains does [³H]-serotonin and therefore seems suitable for use as a photoaffinity labeling probe for serotonin binding sites. Upon irradiation with ultraviolet light, [³H]-NAP-serotonin covalently attaches to protein components of the brain homogenate. Several distinct radioactively labeled proteins have been separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis. Their apparent molecular weights were 80,000, 49,000, and 38,000 (±5%). When 1 μM of unlabeled serotonin or d-lysergic acid diethylamide (d-LSD) was added prior to photolysis, the incorporation of [³H]-NAP-serotonin into these proteins was inhibited significantly. No inhibitory effect was observed when dopamine was used. These observations suggest that the photoaffinity labeled proteins are specific for serotonin binding.     

Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes

The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200-110 to an apparent density of 1.5-2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200-110. cAMP-dependent protein kinase phosphorylated preferentially the 142-kDa band up to a stoichiometry of 0.82 +/- 0.07 (15) mol phosphate/mol peptide. The 56-kDa band was phosphorylated only in substoichiometric amounts. [3H]PN 200-110 bound at 4 degrees C to one site with apparent Kd and Bmax values of 9.3 +/- 1.7 nM and 2.2 +/- 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200-110 in an apparent allosteric manner. (-)Desmethoxyverapamil inhibited the binding of [3H]PN 200-110 at 37 degrees C and stimulated it at 18 degrees C. In agreement with these results, (-)desmethoxyverapamil increased the dissociation rate of [3H]PN 200-110 from 0.29 min-1 to 0.38 min-1 at 37 degrees C and decreased it threefold from 0.046 min-1 to 0.017 min-1 at 18 degrees C. The (+)isomer of desmethoxyverapamil inhibited PN 200-110 binding at all temperatures tested. d-cis-Diltiazem stimulated the binding of [3H]PN 200-110 at 37 degrees C with an apparent EC50 of 1.4 microM and decreased the dissociation rate from 0.29 min-1 to 0.11 min-1. The stimulatory effect of d-cis-diltiazem was temperature-dependent and was seen only at temperatures above 18 degrees C. These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane-bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.