Communicated by James Black, January 6, 1992. ABSTRACT. We have synthesized and charted fluo- rescently labeled dihydropyridines (DHos)as probes forL- ...
Proc. Nati. Acad. Scd. USA Vol. 89, pp. 3586-3590, April 1992 Pharmacology
In vivo labeling of L-type Ca2+ channels by fluorescent dihydropyridines: Evidence for a functional, extracellular heparin-binding site HANS-GUNTHER KNAUS*, THOMAS MOSHAMMER*, KLAUS FRIEDRICH*, HEE CHOL KANGt, RICHARD P. HAUGLANDt, AND HARTMUT GLOSSMANN*t *Institut fur Biochemische Pharmakologie, Peter-Mayr Strasse 1, 6020 Innsbruck, Austria; and tMolecular Probes, Inc., 4849 Pitchford Avenue, Eugene, OR 97402
Communicated by James Black, January 6, 1992
ABSTRACT We have synthesized and charted fluorescently labeled dihydropyridines (DHos) as probes for L-type Ca2+ channels. Racemic as well as (+)- and (-)-1,4-dihydro-
replace radioactive ligands. It was our aim to develop fluorescent DHPs that can largely substitute for radioactive probes in drug-binding assays, to purify Ca2' channels, and to study the L-type Ca2l channel in living cells. We have recently described a high-affinity binding site for heparin on L-type Ca2+ channel, which down-regulates DHP receptors (28). We suggested that the heparin-binding site is located at the extracellular face of the channel, but direct evidence was missing. We report here the properties of fluorescent DHPs that allowed us to study the interaction of heparin with the L-type Ca2l channel in living cells as well as direct labeling of the DHP receptor in purified L-type Ca2+-channel preparations from skeletal muscle.
2,6-dlmethryl4-(2-trifluoromethylphenyl)-3,5-pyrdlhncrboxylic acid 2-(aminoethyl)ethyl ester hydrochlorides were coupled to boron dipyrromethane (Bodipy) derivatives. (4,4-Diluoro5,7-dlmethyl-4-bora-3a,4a-diaza)-3-(s-andaceuepropnadd (DMBodipy)-DHP and (4,4-difluoro-7-styryl-4bora-3a,4adlaza)-3-(s-indacene)propionic acid (STBodipy)-DHP have Kd values in the nanomblar range for membrane-bound or partfally purified skeletal muscle and for neuronal Ltype Ca2+ chanells. (-)- and (+)-STBodpy-DHPs block 4SCa2+ uptake through L-type Ca2+ channels Into GH3 cells with ICY valut-i of 14.8 and 562 nM, respectively. The measurement of bound fluorescence after removal of free DMBodipy-DHP with charcoal shows that the probes can substitute for radioactive lgands to study the properties (equilibrium binding, knetics, aisteric regulation) of partially purified L-ype Ca2 channels from skeletal muscle. L-type Ca2+ channels on GH3 cells were of stereoselectively visualized by using the optical e STBodipy-DHP. Heparin inhibited GH3 cell labeling by (-)STBodipy-DHP with an IC5s value of 9.7 #g/ml and blocked L-type Ca2+-chanemeiated 41Ca2+ iptake with an IC5. value of 32 lag/ml. These argue for an extracellular orientation of the heparin-binding domain of the Ca2+ channel that is coupled to the DHP receptor.
METHODS Synthesi of the (4,4-Dlfluoro-5,7-dmethyl4-bora-3a,4adaz)-3-(s-indacene)proplonlc Acid (DMBodipy)- and (4,4Dfhoro-7-stry-4-bora-3a,4a-diaza)-3-(s-Dee)propioc Acid (STBodipy)-Labeled DHP Enatoers. The pure enantiomers of 1,4-dihydro-2,6-dimethyl4.(2-trifluoromethylphenyl)-3,5-pyridinecarboxylic acid 2-(aminoethyl)ethyl ester were coupled to the fluorescent moieties DMBodipy and STBodipy as described in ref. 21. The structures and the spectral characteristics are shown in Fig. 1. Radloligand Biding. Rabbit skeletal muscle transversetubule or guinea pig-cortex membranes were prepared as described (30). Rabbit skeletal muscle L-type Ca2+ channels were partially purified by solubilization and chromatography on wheat germ agglutinin-Sepharose (31). Fluorescent DHPs and other drugs were diluted and added in dimethyl sulfoxide (32). Protein concentration of membrane-bound Ca2? channels (33) and of partially purified L-type Ca2+ channels (34) was determined as- referenced. 3H-Labeled isopropyl
Radiolabeled dihydropyridines (DHPs) allowed the pharmacological characterization (for reviews, see refs. 1-3), investigation of macroscopic distribution (4), and purification (5) of the L-type Ca2l channels. The ion-conducting a, subunits that carry the DHP-receptor domain from skeletal muscle of different species (6, 7), rabbit smooth muscle, and heart (8-10) have been cloned and expressed (10-12). Visualization of L-type Ca2" channels at the single-cell level has so far only been possible with antibodies (13). Antibody probes, although very useful for distribution studies, can provide only a static view of the channel topography. On the other hand, for neuronal, w-conotoxin GVIA-sensitive Ca2' channels, distribution and lateral mobility were recently investigated with a fluorescent toxin derivative (14), but data on L-type Ca2+ channels are unavailable. For localization studies at the cellular or subcellular level or on living cells fluorescent probes exist for the following receptors: nicotinic acetylcholine (15), dopamine (16, 17), f3-adrenergic (18, 19), benzodiazepine (20), glycine (21), insulin (22), glucagon (23), and vasopressin (24, 25). Fluorescent probes were also developed for voltage-dependent Na' channels (26, 27). In no case, however, have these probes gained general acceptance to
4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5methoxycarbonylpyridine-3-carboxylate [(+)-[3H]PN200110] (85 Ci/mmol; 1 Ci = 37 GBq) was obtained from
Amersham. All receptor-radioligand binding studies were essentially done as described (28). In brief, 0.12-0.31 nM (+)-[3H]PN200-110 was incubated with rabbit skeletal muscle transverse-tubule membrane protein at 0.003-0.008 mg/ml for 60 min at 220C in 0.5 ml of buffer A (50 mM Tris-HCl, pH 7.4/0.1 mM phenylmethylsulfonyl fluoride). For (+)[3H]PN200-110 (0.071-0.121 nM) binding to guinea pig cerebral-cortex membranes (0.125-0.213 mg of protein per ml), Abbreviations: AFU, arbitrary fluorescence units; Bodipy, boron dipyrromethane; DMBOdipy, (4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza)-3-(s-indacene)propionic acid; DHP, dihydropyridine; nH, pseudo-Hill slope; PN200-110, isopropyl 4-(2,1,3-benzoxadiazol4-yl)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonylpyridine-3carboxylate; STBodipy, (4,4-difluoro-7-styryl-4-bora-3a,4a-diaza)3-(s-indacene)propionic acid.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
*To whom reprint requests should be addressed.
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Pharmacology: vi 41
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Proc. Natl. Acad. Sci. USA 89 (1992)
Knaus et al.
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FIG. 1. Structure and spectral characteristics of the fluorescently labeled DHPs DMBodipy-DHP and STBodipy-DHP. All spectra were recorded by using 2 ml of methanol as solvent; the slit width was set to 4 nm (excitation and emission) for all data. -, Absorption spectra of 250 nM DMBodipy-DHP (A) and STBodipy-DHP (B); -, fluorescence emission spectra of 500 nM DMBodipy-DHP (excitation at 488 nm) or STBodipy-DHP (excitation at 546 nm). Structural formulas of DMBodipy-DHP (C) and STBodipy-DHP (D) are shown below.
(0.5 ml of buffer A) were incubated at 370C for 30 min. Separation of bound and free radioligand was done as described (30). Fluorescent Ligand Binding. To determine total and receptor-bound DMBodipy-DHP, a standard curve correlating arbitrary fluorescence units (AFU) with Bodipy-DHP concentrations was constructed in buffer A/0.1% (wt/vol) digitonin/bovine serum albumin at 0.25 mg/ml. We noted that fluorescence in this buffer was more intense than in the organic solvent methanol used for Fig. 1. Dependence of fluorescence intensity on the environment has been reported (19). For kinetic and drug-interaction studies partially purified rabbit skeletal muscle L-type Ca2+-channel protein at 0.003-0.008 mg/ml was incubated with 7.1-25.4 nM DMBodipy-DHP for 60 min at 220C in a final assay volume of 1.5-2 ml (with or without other drugs). For saturation analysis various fluorescent-ligand concentrations were incubated as above. Nonspecific binding was determined with 3,uM
3587
collected by centrifugation and resuspended in buffer B (118 mM NaCl/4.6 mM KCl/1 mM MgCl2/10 mM glucose/5 mM Hepes-NaOH, pH 7.2/0.2 mM CaCl2/0.05% methyl cellulose) and concentrated to 5 x 106 cells per ml. One hundredmicroliter aliquots of this suspension were incubated with the indicated drug concentrations for 5 min at 370C. 45Ca2+ uptake was initiated by adding 1000 gl of depolarization buffer (buffer B/50 mM KCl/72.6 mM NaCI) containing 8-9 AM 45CaC12. Assays were incubated for 2 min and terminated as has been reported (35). Heparin (H-3125, Sigma) concentrations in buffer B were varied, and 45Ca2+ uptake was studied in the presence of 0.5 ,uM (-)-Bay K 8644. Fluorescence Labeling of GH3 Cells. GH3 cells were cultured on 18 x 18-mm lamellae pretreated with 0.1% gelatin (Sigma). GH3 cells were incubated for 2 min in 5 ml of 10 mM Hepes-NaOH, pH 7.4/140 mM NaCl/5 mM KCI/1.2 mM CaCl2/0.5 mM MgCl2/5.5 mM glucose (buffer C) with 5 nM (+)- or (-)-STBodipy-DHP. For definition of nonspecific STBodipy-DHP binding, GH3 cells were preincubated for 10 min at 22°C with 300 nM (+)- or (-)-PN200-110. To study the effects of heparin on (-)-STBodipy-DHP (5 nM)-labeling of GH3 cells, different concentrations of heparin were dissolved in buffer C, and cells were treated as described for the PN200-110 enantiomers. After incubation the lamellae were washed by two 30-sec rinses in ice-cold buffer C/1% bovine serum albumin and an additional rinse in ice-cold buffer C. The slide-mounted GH3 cells were covered with 18 x 18 mm glass lamellae and viewed by using 546/590-nm excitation/ emission dichroic filters and the fluorescence optics of a Zeiss axiophot photomicroscope. Under these light conditions the fluorophore was stable for at least 120 sec. Controls for L-type Ca2+-channel labeling of GH3 cells included omission of STBodipy-DHP, incubation of GH3 cells with 1 ,M of(+)-PN200-110 without fluorescent ligand, or fluorescence staining of Chinese hamster ovary cells CHO-K1 (ATCC CCL 61), which do not express voltage-dependent L-type Ca2+-channels. Under all control conditions no fluorescence signal was seen.
assays
(+)-PN200-110.
After equilibrium (30
min
at
220C, data
not
solution was rapidly cooled to 0-20C. Unbound ligand was separated by adding 50 mg of prewashed charcoal (Merck) suspended in distilled water, followed by centrifugation (5 min, 1PC, 2175 x g, Sorvall RT6000B). Bound ligand was measured in the supernatant with a PerkinElmer LS-50 luminescence spectrometer (excitation wavelength 488 nm at a slit width of 4 nm; emission was recorded at 517 nm at a slit width of 20 nm). 45Ca2 Uptake into GH3 Cells. 45CaC12 (20-40 mCi/mg) was from Amersham. GH3 cells (ATCC no. CCL 82.1) were grown in spinner culture in Dulbecco's minimal essential medium/ 17.5% (vol/vol) fetal calf serum (GIBCO)/streptomycin at 0.2 mg/ml/penicillin at 50 units/ml in 92% 02/8% CO2. Cells were shown) the
assay
RESULTS Boron Dipyrromethane (Bodipy)-Labeled DHPs Bind with High Affinity to L-Type Ca2+ Channels and Are Biologically Active. The interactions of DMBodipy-DHP and STBodipyDHP with L-type Ca2+-channel-associated drug receptors were assessed by determining their ability to inhibit specific (+)-[3H]PN200-110 binding. The fluorescent DHPs recognized neuronal L-type Ca2+ channels with higher affinity than those in skeletal muscle. (-)-DMBodipy-DHP and (-)STBodipy-DHP had K, values for neuronal Ca2+ channels of 0.90 ± 0.04 nM and 0.90 ± 0.13 nM (n = 3), respectively. For skeletal muscle Ki values of 4.88 ± 2.64 nM and 2.25 ± 1.20 nM (n = 3) were determined. Discrimination of the optical antipodes was more pronounced for STBodipy-DHP, as the (+) enantiomer had a K, value of 26.3 ± 8.4 nM for neuronal and 100.6 ± 38.4 nM for skeletal muscle Ca2+ channels. In contrast, (+)-DMBodipy-DHP bound with a K1 value of 2.8 ± 0.7 nM (neuronal Ca2+ channels) and 12.5 ± 2.6 nM (skeletal muscle Ca2+ channels). Because the eudismic ratio for the STBodipy-DHPs was between 29 and 45 this label was selected for stereoselective fluorescence labeling of L-type Ca2+ channels on GH3 cells (see below). The enantiomers of STBodipy-DHP are functional Ca2+-channel blockers. Concentration-dependent block of depolarization-induced45Ca2+ uptake of GH3 cells occurred with an IC50 of 562 ± 72 nM [(+)-STBodipy-DHP] and 14.8 ± 4.3 nM [(-)-STBodipyDHP], respectively. Under these conditions, the high-affinity DHP (+)-PN200-110 inhibited with an IC50 value of 1.62 + 0.78 nM. In Vitro Binding Studies with Fluorescently Labeled DHPs. To demonstrate the use of the Bodipy-labeled DHPs to
Proc. Natl. Acad. Sci. USA 89 (1992)
Pharmacology: Knaus et al. characterize L-type Ca2l channels directly, we selected
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concentration-dependent decrease of receptor-bound DMBodipy-DHP concentration) is shown in Fig. 2C. In Vivo Visualization of L-Type Ca2+ Channels of GH3 Cells. L-type Ca2+ channels of GH3 cells were visualized in vivo using the enantiomers of STBodipy-DHP under exactly the same conditions and at identical concentrations (Fig. 3). The rationale behind this approach was that the optical antipodes have identical physicochemical properties (including fluorescence parameters and lipid solubilities) but were well discriminated by the DHP-receptor domain on the a1 subunit with eudismic ratios between 39 (biological activity) and 29 (Ki values for neuronal Ca2' channels). The eutomer, (-)STBodipy-DHP, stained GH3 cells with much brighter fluorescence than the distomer, (+)-STBodipy-DHP. Moreover, using the optical antipodes of PN200-110, we can show the stereoselective inhibition of STBodipy-DHP labeling; this result provides additional proof for the L-type Ca2+channel interaction of our fluorescence ligands. Heparin Blocks L-Type Ca2+ Channel Labeling by (-)STBodipy-DHP and 4SCa2 Inflsux in GH3 Cells. Heparin inhibits DHP labeling of membrane-bound and purified L-type Ca2+ channels from skeletal muscle and of membranebound L-type Ca2+ channels in heart and neuronal membranes (28). In functional experiments heparin increased the current through L-type Ca2+ channels of guinea pig cardiac myocytes (28). We therefore suggested that the heparinbinding domain is located at the extracellular side of the Ca2+ channel, but direct evidence by labeling intact cells was missing. Our fluorescent probes offered the opportunity to apply heparin, which cannot permeate the plasma membrane, to GH3 cells labeled with (-)-STBodipy-DHP. Heparin inhibited the labeling with an IC50 value of -9.7 ,ug/ml; 45Ca2+ uptake in the presence of the Ca2+-channel-activating DHP Bay K 8644 was inhibited with an IC50 value of =31.7 ,g/ml (Fig. 4).
DMBodipy-DHP because it has a more intense fluorescence than STBodipy-DHP (see Fig. 1). In Fig. 2 we show examples for equilibrium saturation analysis, kinetics, as well as drug-interaction studies, using partially purified L-type Ca2" channels from rabbit skeletal muscle. These preparations have an average density of 286 pmol/mg of protein, labeled by the standard radioligand (+)-[3H]PN200-110 (36). When increased concentrations of (-)-DMBodipy-DHP were incubated with the partially purified preparation, a Kd of 29.4 nM and a maximum density of binding sites of 284.9 pmol/mg of protein was found (Fig. 2A). The signal-to-noise ratio was excellent, as shown by the very small difference between total and specific (i.e., receptor-bound) concentrations of fluorescent ligand. The Kd value for (-)-DMBodipy-DHP is 5.3-fold higher than that for (+)-PN200-110 (5.5 nM, see ref. 36). Thus, (-)-DMBodipy-DHP reflects the characteristic loss in affinity seen for DHPs when Ca2+ channels from skeletal muscle are solubilized and partially purified by wheat germ agglutinin-affinity chromatography (36). By incubating Bodipy-DHPs with transverse-tubule membranes from skeletal muscle we could isolate L-type Ca2+ channels up to the sucrose-density gradient step (31) by measuring fluorescence (data not shown). The Kd for (-)-DMBodipy-DHP for partially purified L-type Ca2+ channels was 0.128 min-' (Fig. 2B), whereas it was 0.03 min-1 for (+)-[3H]PN200-110 at 220C (data not shown). Clearly, the lower affinity of (-)DMBodipy-DHP compared with (+)-PN200-110 is mainly from an increase of the dissociation rate constant, kL1. The DHP-receptor domain on the a1 subunit is coupled through reciprocal allosteric mechanisms to other receptors and to Ca2+-binding sites (1, 37). Fig. 2C exemplifies for the (socalled) positive regulators (+)-cis-diltiazem, (+)-tetrandrine, and (-)-BM 20.1140 [ethyl 2,2-diphenyl-4-(1-pyrrolidino)-5(2-picolyl)oxyvalerate] (38) that our fluorescent label in the presence of these drugs is behaving like a typical DHP Ca2+ antagonist: radiolabeled DHPs (at ligand concentrations that do not saturate the receptor) increase their equilibrium binding, reflecting a decrease in Kd (38). Another class of drugs, the phenylalkylamines, can act as positive or negative regulators of DHP binding (31). The negative heterotropic allosteric effects of (+)-desmethoxyverapamil (as reflected by a X
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FIG. 2. In vitro labeling of L-type Ca2+ channels by DMBodipy-DHP. (A) Saturation analysis. (-)-DMBodipy-DHP (range 3.25-78.15 nM) was incubated with partially purified Ca2+-channel protein at 0.00345 mg/ml for 20 min at 22°C in a final assay volume of 2 ml. o, Total binding; *, specific binding, defined by total binding minus nonspecific binding in the presence of 3 ,uM (-+-)-PN200-110. Data are means from duplicates , mean + asymptote SD) with the following that differed, on average, by 3.7%. Data points for specific binding were computer-fitted ( parameters: Kd, 29.4 + 2.5 nM; Bmax, 0.985 ± 0.04 nM; this corresponds to 285 pmol of DHP-binding sites per mg of protein. (B) Kinetics. (-)-DMBodipy-DHP (20.58 nM) was incubated with Ca2+-channel protein at 0.017 mg/ml in 1.5 ml until equilibrium (22°C for 20 min; the specifically bound ligand at equilibrium was 0.82 nM, corresponding to 22.3 AFU). Receptor-ligand complex decay was initiated by adding 3 ,uM (+)-PN200-110. kL1 was 0.128 ± 0.009 min-' (-, monoexponential decay). Means from duplicate determinations are shown. (C) Drug-interaction profile. Partially purified rabbit skeletal muscle L-type Ca2+-channel protein (0.004 mg/ml) was incubated with 8.5 nM DMBodipy-DHP without (control binding) and with increased concentrations of various drugs as indicated. Specifically bound ligand at equilibrium was between 0.25 and 0.36 nM. *, (+)-PN200-110 [IC50 of 3.89 ± 0.41 nM, pseudo-Hill slope (nH) = 1.21 + 0.11]; o, (-)-PN200-110 (IC50 of 263 ± 11 nM, nH = 1.28 + 0.31); *, (+)-cis-diltiazem (EC50 of 487 ± 31 nM, nH = 1.57 ± 0.14, maximal stimulation to 194 ± 8%); O, (-)-cis-diltiazem (IC50 of 74.13 ± 1.3 ,uM, nH = 0.86 ± 0.07); o, (±)desmethoxyverapamil (IC50 of 5.49 ± 0.2 AM, nH = 0-99 + 0.09); A, (+)-tetrandrine (EC50 of 131 ± 4 nM, nH = 1.13 + 0.14, maximal stimulation to 269 ± 11%); A, (-)-BM 20.1140 [ethyl-2,2-diphenyl-4-(1pyrrolidino)-5-(2-picolyl)oxyvalerate] (EC50 of 457 ± 21 nM, nH = 0.88 + 0.07, maximal stimulation to 161.5 ± 4.5%).
Pharmacology: Knaus et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
3589
FIG. 3. Visualization of L-type Ca2+ channels on GH3 cells. (A-F) Emitted STBodipy-DHP fluorescence with 546/590-nm excitationemission dichroic filters. (Insets) Normal illumination. Fluorescence of clearly outlined cells was measured by photon counting. Fluorescence staining of slide-grown GH3 cells incubated with 5 nM (-)-STBodipy (A, B, and C) or 5 nM (+)-STBodipy-DHP (D, E, and F). (A) (-)-STBodipy-DHP fluorescence (control, normalized to 100o fluorescence, n = 18 cells). (B) Conditions were similar to those in A except that 300 nM (+)-PN200-110 was present (21.3 ± 6.1% of control fluorescence, n = 46 cells). (C) Conditions were similar to those in A except that 300 nM (-)-PN200-110 was present (91.0 ± 7.5% of control fluorescence, n = 12 cells). (D) (+)-STBodipy fluorescence (11.3 ± 3.3% of control fluorescence, n = 38 cells). (E) Conditions were similar to those in D except that 300 nM (+)-PN200-110 was present (6.3 ± 0.7% of control fluorescence, n = 17 cells). (F) Conditions were similar to those in D except that 300 nM (-)-PN200-110 was present (6.7 ± 0.9%o of control fluorescence, n = 19 cells). Note that (+)-PN200-110 is the more active enantiomer on L-type Ca2+ channels and (-)-PN200-110 is much less active (1, 2), whereas (-)-STBodipy-DHP is of higher affinity than (+)-STBodipy-DHP (see text). (x320.)
channel a1 subunits in solubilized preparations, and to study their kinetics by simply measuring fluorescence. We also successfully used a prelabeling protocol to isolate the Ca2l channel from skeletal muscle transverse-tubule membranes. Thus, our labels can substitute for radioactive ligands in almost any aspect. One disadvantage is the somewhat lower affinity [e.g., (-)-DMBodipy-DHP had a K, value of 4.88 nM for particulate skeletal muscle Ca2+ channels] compared with the newer generation of DHPs-e.g., (-)-[3H]azidopine (39), [35S]sadopine (40), (-)-[125 ]iodipine (41), or (+)-[3H]PN200110 (28). However, these binding constants for the fluorescent DHPs are in the same range as those of the first generation DHPs-e.g., [3H]nifedipine (Kd = 4.9 nM), [3H]nimodipine (Kd = 3.6 nM), or [3H]nitrendipine (Kd = 3.6 nM) (42).
The availability of pairs of fluorescent optical antipodes with favorable affinity ratios for receptor binding greatly helped to show without doubt (as proven by stereospecific labeling and stereospecific inhibition) that the DHP-receptor domain of the L-type Ca2" channel is labeled by these ligands in vivo. To obtain more direct proof for the suggested extracellular location of the heparin-binding domain (28) we monitored (-)-STBodipy fluorescence on living GH3 cells with increased concentrations of heparin. The heparinbinding site of the L-type Ca2' channel is coupled in a negative heterotropic allosteric manner to the DHP receptor (28). If this site faces the cytosol, heparin should not impair fluorescent labeling of the GH3 cells by (-)-STBodipy-DHP. Furthermore, a cytosolic orientation would exclude any heparin effects on the DHP agonist-induced 45Ca2+ uptake
Pharmacology:
3590
Knaus et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 4. Inhibition of (-)-STBodipy-DHP fluorescence and 45Ca2+ uptake of GH3 cells by heparin. (A) Heparin inhibition of (-)-STBodipy-DHP fluorescence of GH3 cells. Fluorescence was quantitated as above. Each data point is the mean of 18-42 cells. Data are normalized to fluorescence without heparin and were fitted as described (28) with the following optimized parameters: IC50 = 9.31 1.3 ,ug/ml; nH was 0.86 0.19. (B) Heparin inhibition of 45Ca2+ uptake with 0.5 ,uM (-)-Bay K 8644. Data points are means from two independent experiments done in duplicate. Non-L-type Ca2+-channel-mediated uptake was defined by 1 ,uM (+)-PN200-110 and was subtracted to yield 45Ca2` uptake through L-type Ca2+ channels (100%). IC50 = 31.7 3.2 ,ug/ml; nH was 0.97 0.2. ±
17.
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because the highly charged anion is not expected to cross the plasma membrane. To our satisfaction heparin displaced the fluorescent DHP and blocked 45Ca2+ uptake, supporting our suggestion that the heparin-binding domain of the L-type Ca2+ channel is on the extracellular face. Interestingly, the inhibitory action of heparin on L-type Ca2+ channels in GH3 cells and its previously reported augmentation of Ca2+ currents in cardiac myocytes (28) are shared by an endogenous low-molecular-weight peptide that is highly acidic and inhibits DHP binding in a way similar to that of heparin (29). If this peptide and heparin share the same binding site, a physiological role for the former is likely. This work was supported by Fonds zur Forderung der Wissenschaftlichen Forschung Grants S4501-MED and S4502-MED (to H.G.), by a grant from the Bundesministerium fur Wissenschaft und Forschung (to H.G.), by the Dr. Legerlotz Foundation (to H.-G.K.) and by National Institutes of Health Grant GM-37347 (to R.P.H.). 1. Glossmann, H. & Striessnig, J. (1988) Vitam. Horm. (N. Y.) 44, 155-328. 2. Glossmann, H. & Striessnig, J. (1990) Rev. Physiol. Biochem. Pharmacol. 114, 1-105. 3. Hosey, M. M. & Lazdunski, M. (1988) J. Membr. Biol. 104, 81-105. 4. Cortds, R., Supavilai, P., Karobath, M. & Palacios, J. M. (1984) J. Neural Transm. 60, 169-197. 5. Curtis, B. M. & Catterall, W. A. (1984) Biochemistry 23, 21132118. 6. Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1987) Nature (London) 328, 313-318. 7. Grabner, M., Friedrich, K., Knaus, H. G., Striessnig, J., Scheffauer, F., Staudinger, R., Koch, W. J., Schwartz, A. & Glossmann, H. (1991) Proc. Natl. Acad. Sci. USA 88,727-731. 8. Biel, M., Ruth, P., Bosse, E., Hullin, R., Stuehmer, W., Flockerzi, V. & Hofmann, F. (1990) FEBS Lett. 269, 409-412. 9. Koch, W. J., Ellinor, P. T. & Schwartz, A. (1990) J. Biol. Chem. 265, 17786-17791. 10. Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y.,
20. 21. 22. 23.
24. 25.
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42.
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