Identification of ω-Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.     

Specific high affinity binding of the calcium channel antagonist, [3H]nitrendipine, to rat liver microsomes

Some key properties of the binding of [3H]nitrendipine, an analogue of the 1,4-dihydropyridine, nifedipine, to a plasma membrane enriched microsomal fraction from the rat liver are described. Specific binding was saturable, linear with protein concentration, and reversible. The apparent equilibrium dissociation constant, KD, was 4.20 +/- 0.22 nM and the maximum density of binding, Bmax, was 3.02 +/- 0.17 pmol/mg of protein determined from Scatchard analysis of binding at 10 degrees C. Inhibition of binding was specific for dihydropyridines with competitive inhibition being noted with nifedipine and 4-chloronifedipine, as well as BAY K-8644, a calcium channel agonist. A biphasic displacement curve was recorded for methoxy verapamil (D-600), and a triphasic competition curve with lanthanum (La3+), and diltiazem demonstrated competitive kinetics. The high affinity binding site for nitrendipine in the liver, although having some similar properties to those sites described in skeletal muscle, would appear to be distinctive with respect to its unique sensitivity to D-600 and diltiazem. We speculate that this binding site may represent a Ca2+ channel responsible for regulating Ca2+ influx and hepatic glycogenolysis.     

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.     

Effects of Ca2+ Channel Blockers on Ca2+ Translocation Across Synaptosomal Membranes

The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.     

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.     

3H]nitrendipine binding to adrenal capsular membranes

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. We therefore studied the [3H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with KD = .26 +/- .04 nM and Bmax = 105 +/- 5.7 fmol/mg protein. Specific binding of [3H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine much greater than verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [3H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production.     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

(+)-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)     

Partial purification of a protein from maize (Zea mays) coleoptile membranes binding the Ca2+-channel antagonist verapamil.

A protein that binds the calcium-channel antagonist verapamil has been partially purified from maize (Zea mays) coleoptile membranes. The protein was solubilized with the detergent CHAPS ([ 3-(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate) and purified by a combination of ion-exchange, gel-filtration and hydrophobic-interaction chromatography. This resulted in a 120-fold purification. SDS/polyacrylamide-gel-electrophoretic analysis of the polypeptides from the final purification step indicated that the verapamil-binding protein may have a major component of Mr 169,000. The dissociation constants for specific binding of [3H]verapamil to crude and CHAPS-solubilized maize coleoptile membrane fractions are 72 nM and 158 nM respectively, with respective binding-site concentrations of 135 pmol/mg of protein and 78 pmol/mg of protein. In both cases the Scatchard plots are linear, indicating a single class of binding sites. [3H]Verapamil binding to crude maize coleoptile membrane fractions could not be displaced by unlabelled desmethoxyverapamil or by nifedipine, but could be displaced by unlabelled methoxyverapamil.     

Dihydropyridine Binding Sites Regulate Calcium Influx Through Specific Voltage-Sensitive Calcium Channels in Cerebellar Granule Cells

In primary cultures of cerebellar granule cells, [3H]nitrendipine binds with high affinity to a single site (KD 1 nM and Bmax 20 fmol/mg protein). The 1,4-dihydropyridine (DHP) class of compounds such as nitrendipine, nifedipine, and BAY K 8644 displace [3H]nitrendipine binding at nanomolar concentrations. Verapamil partially inhibits whereas diltiazem slightly increases the [3H]nitrendipine binding. In these cells, the calcium influx that is induced by depolarization is very rapid and is blocked by micromolar concentrations of inorganic calcium blockers such as cadmium, cobalt, and manganese. The calcium influx resulting from cell depolarization is potentiated by BAY K 8644 and partially inhibited (approximately 40%) by nitrendipine and nifedipine. Other non-DHP voltage-sensitive calcium channel (VSCC) antagonists, such as verapamil and diltiazem, completely blocked the depolarization-induced calcium influx. This suggested that nitrendipine and nifedipine block only a certain population of VSCCs. In contrast, verapamil and diltiazem do not appear to be selective and block all of VSCCs. Perhaps some VSCCs can be allosterically modulated by the binding site for the DHPs, whereas verapamil and diltiazem may block completely the function of all VSCCs by occupying a site that differs from the DHP binding site.     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

3H]nitrendipine receptors in skeletal muscle

The richest source of receptors for the organic calcium channel blocker [3H]nitrendipine in muscle is the transverse tubule membrane. The tubular membrane preparation binds [3H]nitrendipine with a high affinity and has a very high number of [3H]nitrendipine binding sites. For example, for the transverse tubule membrane preparation from rabbit muscle, the dissociation constant of the nitrendipine-receptor complex is 1.8 +/- 0.3 nM and the maximum binding capacity Bmax = 50 +/- 6 pmol/mg of protein. Similar results have been found with a membrane preparation from frog muscle. The dissociation constant found at equilibrium is near that determined from the ratio of rate constants for association (kappa 1) and dissociation (kappa-1). Binding of [3H] nitrendipine is pH-dependent and reveals the presence of an essential ionizable group with a pK of 5.4 on the nitrendipine receptor. The binding is destroyed by proteases showing that the receptor is a protein. Three different classes of Ca2+ channel blockers inhibit [3H]nitrendipine to its specific site. (i) The dihydropyridine analogs of nitrendipine which are competitive inhibitors of [3H]nitrendipine. These molecules form tight complexes with the nitrendipine receptor with dissociation constants between 1.4 and 4.0 nM. (ii) Other antiarrhythmic molecules like verapamil, amiodarone, bepridil, and F13004 which are noncompetitive inhibitors of [3H]nitrendipine binding with dissociation constants between 0.2 and 1 microM. (iii) Divalent cations like Ni2+, Co2+, Mn2+, or Ca2+ which are noncompetitive inhibitors of [3H]nitrendipine binding with the following rank order of potency: Ni+ (K0.5 = 1.8 mM) greater than Co2+ (K0.5 = 2.7 mM) greater than Mn2+ (K0.5 = 4.8 mM) greater than Ca2+ (K0.5 = 65 mM).     

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.     

(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle

(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.     

Binding Ca2+ to intracellular or to extracellular sites of dihydropyridine receptor of rabbit skeletal muscle discriminates between in vitro binding of Ca2+-channel agonist and antagonist

Transverse tubule membrane vesicles contain dihydropyridine receptor of rabbit skeletal muscle in an insideout orientation. Digitonin-solubilized, purified dihydropyridine receptor is embedded in digitonin vesicles in an outside-out orientation. Ca2+ selectively stimulates binding of the Ca2+-channel antagonist [3H]PN200-110 to dihydropyridine receptor in the outside-out but not the inside-out orientation. The dissociation constant for binding Ca2+ to the extracellular Ca2+-specific binding site of dihydropyridine receptor is 2-3 microM. The data demonstrate that binding Ca2+ to the extracellular high-affinity Ca2+-binding site is required for binding dihydropyridines to dihydropyridine receptor. This binding is inhibited, however, by 1-10 mM concentrations of any divalent cation tested (Ba2+, Mn2+, Mg2+). Also, Ca2+ selectively stimulates binding of the Ca2+-channel agonist [3H]BayK8644 to dihydropyridine receptor in the inside-out orientation. The titration of this Ca2+ dependence indicates that the dissociation constant for binding Ca2+ to the intracellular Ca2+-specific binding site of dihydropyridine receptor is in the millimolar range. Thus, binding Ca2+-channel agonist or antagonist to dihydropyridine receptor is modulated by binding Ca2+ to different sites of the receptor. Measurements of dissociation rate constants for binding [3H]PN200-110 to dihydropyridine receptor in the presence of diltiazem, verapamil and/or Ca2+ indicate that Ca2+, like diltiazem or verapamil, is an allosteric effector of this receptor.     

Kainate Receptors in Xenopus Central Nervous System: Solubilisation with n-Octyl-β-d-Glucopyranoside

[3H]Kainate binding to membrane homogenates and detergent extracts prepared from Xenopus central nervous system was evaluated in 50 mM Tris-citrate buffer, pH 7.0. In membrane fragment preparations, [3H]kainate bound with a KD of 54.4 nM to a large number of sites (Bmax = 27.8 pmol/mg of protein). Up to 80% of the total number of membrane-bound binding sites were solubilised using the nonionic detergent n-octyl-beta-D-glucopyranoside. Values for the KD of [3H]kainate for solubilised binding sites were 46.0 nM and 53.6 nM derived from equilibrium and kinetic binding experiments, respectively. Competitive binding studies revealed that a variety of ligands had similar Ki values in both membranes and solubilised extracts, with domoate and kainate being the most potent inhibitors of [3H]kainate binding. The dissociation rate of [3H]kainate from solubilised binding sites was 0.022 min-1. The binding component migrated in sucrose density gradients in a single 8.6S peak. These results demonstrate that the kainate receptor in Xenopus central nervous system, although similar to the [3H]kainate binding site from goldfish brain, differs in a number of important respects. In particular, the slower dissociation rate and higher affinity of [3H]kainate suggest that Xenopus provides the most convenient model system yet investigated for biochemical analysis of kainate receptors.     

Evidence for a Single Naphthylphthalamic Acid Binding Site on the Zucchini Plasma Membrane

The binding of [2,3,4,5,(n)-3H]N-1-napthylphthalamicacid ([3H]-NPA) to zucchini (Cucurbita pepo L.) plasma membranes was examined in detail using two different filtration assays and the results were rigorously analyzed by saturation curves, double-reciprocal plots, Scatchard plots, Hill plots, and the computer program Ligand (P.J. Munson, D. Rodbard [1980] Anal Biochem 107: 220-239). To facilitate these analyses, a new assay that allows rapid and quantitative analysis of [3H]NPA binding with high reproducibility and ease of manipulation has been developed. These detailed kinetic analyses indicate that only one binding site for [3H]NPA (Kd = 16 nM) was associated with the zucchini plasma membrane. Analysis of [3H]NPA dissociation by several auxin transport inhibitors revealed similar dissociation constants with both plasma and microsomal membrane. Collectively, these data indicate the presence of only one binding site for NPA associated with the zucchini plasma membrane.     

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)     

Diltiazem potentiates the negative inotropic action of nimodipine in heart

In Langendorff perfused rat hearts, nimodipine enhances coronary flow and inhibits contractility. The binding of [3H]nimodipine (160 Ci/mmol) to sarcolemma isolated from dog heart revealed a KD of 0.2 nM. d-cis-Diltiazem, but not 1-cis-diltiazem, a less active stereoisomer, stimulated [3H]nimodipine (0.17 nM) binding to sarcolemmal membranes (ED50 for diltiazem = 1.1 microM). In the presence of 10 microM d-cis-diltiazem, [3H]nimodipine binding sites were doubled, but there was no change in the apparent affinity. Perfused rat hearts were treated with 250 nM d-cis-diltiazem. The negative inotropic response to nimodipine was dramatically potentiated (I50, from 1.1 to 0.033 microM). The pharmacological and binding effects were observed only at 37 degrees C. It is possible that diltiazem in some way converts low affinity to high affinity sites.