THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U S.A. Vol. 257, No. 15, Issue of August IO. pp. X964-8971. 19x2

Binding of Quinacrine, a Fluorescent Local Anesthetic Probe, to Mammalian Axonal Membranes EVIDENCE FOR A LOCAL ANESTHETIC RECEPTOR SITE"

(Received for publication, March 22, 1982)

Martin Greenberg and Tian Yow Tsong From the Department of Physiological Chemistry, The Johns Hopkins UniversitJl School of Medicine, Baltimore, MaJyland 21205

The fluorescence probe quinacrine was found to bind specifically to the axonal membrane fraction prepared from bovine corpus callosum. The fluorescent intensity of bound quinacrine was enhanced 20 to 25% compared to free quinacrine, and there was also a blue shift of the emission maximum from 500 to 492 nm. Quinacrine binding was of high affinity with an apparent KD of 0.6 to 1.2 p ~ , and the density of binding sites was about 5 nmol/mg of protein depending on membrane prepara- tions. This high affinity binding was lacking in lipid extract of axonal membranes nor was it detected in myelin membranes prepared concomitantly with ax- onal membranes. The high affinity quinacrine binding was found preferentially in nervous tissues versus non- nervous tissues.

Local anesthetics, such as dibucaine, dimethisoquin, tetracaine, etidocaine, lidocaine, and procaine, com- peted for the quinacrine binding in axonal membranes. The effectiveness of this competition, e.g. IC5os (the concentration of anesthetics to block 50% of quinacrine binding) correlated linearly with the potency of the local anesthetics to block the axonal conduction of desheathed frog sciatic nerves (Ch0s, concentration for 50% blockage) in the anesthetic concentration range of 10" to IOL2 M. Quinacrine was also shown to be a potent inhibitor of the axonal conduction. We found that Csoa of quinacrine upon axonal conduction had an apparent dependence on the nerve-stimulating voltage, e.g. CEO% of 0.2 PM was measured at 240 mV stimulation voltage and 10 PM was measured at 300 mV.

The high affinity quinacrine binding sites are judged to be protein in nature. First, energy transfer was detected between tryptophan fluorescence of mem- brane proteins and the bound quinacrine. Second, 90 to 95% of the quinacrine binding was abolished by trypsin treatment; moreover, treatment with phospholipases C and D did not show significant reduction in quinacrine binding.

The data presented here indicate the existence of a local anesthetic receptor site in mammalian axonal membranes. Quinacrine appears to be a potent anes- thetic probe for the receptor and should be useful for studying local anesthetic function in other nervous sys- tems as well.

Local anesthetics affect neural conduction, both at the

* This work was supported by National Institutes of Health Grant GM 28795. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

synapse and along the axon, but the mechanism of their action remains to be elucidated (1-3). Various investigators have presented evidence favoring either protein or lipid as sites of action. Pringle and Miller (4) have argued that local anes- thetics act by fluidizing the lipid bilayer. Other investigators have shown that local anesthetics affect the gel-to-liquid crystalline phase transition temperature of model membrane lipids (5-8) and increase the bilayer permeability to ions and molecular probes (7). In contrast, Narahashi (9, 10) and Hille (11) have implicated the sodium channel as a major site of local anesthetic action in squid axon. At the neuromuscular junction, Ruff (12, 13), Adams and Feltz (14, 15), and Stein- bach (16) have likewise implicated the ionophore associated with the acetylcholine receptor,

The development of quinacrine as a fluorescence probe has provided a convenient tool for studyinglocal anesthetic action. Quinacrine mimics local anesthetics by inhibiting noncompet- itively the electrophysiological response of electroplaque prep- arations to acetylcholine, and in membrane homogenates, quinacrine binding is displaced by clinically useful local an- esthetics (17). The pharmacology of quinacrine binding has supported the notion that, in electroplaque, local anesthetics bind to the acetylcholine receptor ionophore (15,18,19). Also, competition for binding between quinacrine and histrionico- toxin, a toxin believed to block the ionophore, further supports this idea (20, 21). Recently, quinacrine has been shown to block neuromuscular condition in mammalian synapses at fairly low (5-10 pM) drug concentrations (22).

Since local anesthetics also affect the propagation of neu- ronal action potentials, it is desirable to study quinacrine binding in a synapse-free axonal preparation. Recently, De- Vries and collaborators have prepared a membrane fraction derived from bovine corpus callosum, a rich source of axons, that is highly enriched for axonal membranes (23-25). These axonal membranes are well characterized (23); they are syn- apse free (24, 25), devoid of myelin (23) and other membrane contaminants from astrocytes (26) or oligodendrocytes (23), and are highly enriched (23) in specific binding of excitable sodium channel probes (tetrodotoxin and saxitoxin) and spe- cific activity of enzymes known to be associated with plasma membranes ((Na'/K')-ATPase and acetylcholinesterase). We report here a study of quinacrine binding to this preparation by fluorescence spectroscopy. Our results indicate that quin- acrine competes with local anesthetics for binding and that it appears to have a specific protein binding site. We also report here the effects of quinacrine on axonal conduction in frog sciatic nerve.

EXPERIMENTAL PROCEDURES

Materials-Quinacrine, dibucaine, lidocaine, procaine, and tetra- caine hydrochlorides, tetraethylammonium, trypsin (Type I), tetro-

8964

dotoxin, pronase (Streptomyces griseus Type V), a-chymotrypsin (Type II), phospholipase Al (lecithinase A, bee venom), phospholi- pase c (lecithinase type I, CZostridium welchii), phospholipase D (Type I, cabbage), ribonuclease A (Type XII-A, bovine pancreas), deoxyribonuclease I (DN-25, bovine pancreas), trasylol (aprotinin), and phenylmethylsulfonylfluoride were purchased from Sigma. 3,4- Diwinopyridine and D20 were from Aldrich. Etidocaine and prilo- caine hydrochlorides were from Astra Pharmaceutical (Worster). Quotane or dimethisoquin hydrochloride was from Smith, Kline and French (Philadelphia, PA). Bupivicaine hydrochloride was from Ster- ling-Winthrop Research Institute (Rensselaer, NY). Barbiturates in sodium salt form were obtained from Johns Hopkins Hospital. a- Flupenthixol, @-flupenthixol, thioridazine, haloperidol, chlorproma- zine, fluphenazine, imipramine, promethazine, and mesoridazine were kindly provided by Dr. Solomon H. Snyder, Department of Neuro- sciences, Johns Hopkins University School of Medicine. Jumbo male bullfrogs (Rana catesbiana) were supplied from Parco Scientific, Vienna, Ohio.

Membrane Isolation-Axonal membranes and myelin membranes were prepared from fresh or frozen bovine corpus callosum according to the method of DeVries (23, 24) except trasylol (20 units/ml) and phenylmethylsulfonylfluoride (0.1 mM) were present during the initial homogenization steps. The membrane fractions were collected and resuspended at a 0.5-1.0 m g / d of protein concentration in Hanks' medium (NaCI, 137 mM; KC1, 5.36 m; NaHCO.3, 4.17 mM; KHaPO,, 0.4 mM; Na2HP04, 0.4 mM; MgCL * 6H20, 0.7 m; MgSOr. 7H20, 0.4 mM; CaCI2, 1.26 IPM; NaNz, 0.02% w/v; pH 7.2 to 7.4 and omitting glucose and phenol red). Membrane protein was determined by fluorescamine (27) or Bio-Rad assay using bovine serum albumin as standard. A typically consistent yield of axonal membranes was 3-4 mg of protein per 12 g wet weight of corpus callosum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Na'/K' ATPase, and ac- etylcholinesterase assays were performed on the axonal and myelin preparations as described by DeVries et al. (23, 28), and the results were consistent with their data. The membrane fractions were stored in aliquots a t -80 "C and used within 2 weeks of preparation.

In addition, a fresh bovine brain was quickly dissected on ice into individual brain regions and stored frozen in plastic vials a t -80 "C until use. Brain regions were finely minced, homogenized a t 4 "C (Waring blender, high speed, 60 s) in 100 volumes of Hanks' medium plus 20 units/ml of trasylol and 0.1 mM phenylmethylsulfonylfluoride, and centrifuged at 50,000 X g for 30 min (33, 34). The pellets were resuspended in the same volume of fresh buffer, centrifuged as before, and the final pellets resuspended in Hanks' buffer for protein deter- mination (as before) and quinacrine binding studies.

Fresh bovine organs were also obtained concomitantly with the whole brain (all from a local slaughterhouse). Membrane pellets were prepared and resuspended as described above, except that the initial homogenates were passed through a layer of cheesecloth to remove connective tissue debris.

Fluorescence Measurements-All measurements were performed in an Aminco-Bowman spectrofluorimeter thermostatted a t 23 "C. Quartz fluorescence cells with a cross-section (10 X 10 mm) were filled with 1.5 ml of sample and reference solutions. There was no problem of membrane settling during the course of the experiments. The fluorescent signal upon quinacrine binding was stable for hours.

We performed two types of fluorescence measurements. First, we followed the fluorescence enhancement of quinacrine upon binding to axonal membrane preparations (increase in quantum yield, emission spectral shift) (Set A). Second, we studied the quenching of trypto- phan fluorescence of the axonal membrane preparation upon quina- crine binding (Set B).

In Set A the excitation wavelength was 350 nm, the emission wavelength was 500 nm, and the slit width was 1 nm. In all experi- ments the background signal (light scattering and fluorescence) was first recorded and appeared to be negligible. The emission spectra were recorded on a Sargent recorder, and the slit width was adjusted to 1 nm. An excitation wavelength of 350 nm was chosen because it is an isobestic point in the pH-dependent absorption spectrum of quinacrine (29). Absorbance values at 350 nm were less than 0.1, obviating interference from an inner filter effect. Under Set A condi- tions the fluorescence intensity was linear with quinacrine concentra- tion in the range 0.1 to 20 WM. From 20 to 100 p~ concentrations the fluorescence intensity began to quench, probably due to aggregation of quinacrine in solution (29). The fluorescence enhancement, called h F , was the difference in fluorescence intensity of quinacrine in the presence of membranes and that in the absence of membranes. The fluorescence enhancement was linear with axonal membrane protein

concentration from 5.0 to 100 pg/ml. Storing the axonal membranes at 4 "c for 2 weeks or treating by repeated freezehhaw cycles caused a marked drop in binding of quinacrine.

In Set B the excitation wavelength was 290 nm, the emission wavelength was 330 nm, and the slit width was I nm. We chose an excitation wavelength of 290 nm for it is also at a pH isobestic point for quinacrine absorption (29). The binding of quinacrine to axonal membranes was followed by the decrease of tryptophan fluorescence emission. The concentrations of quinacrine used were kept low, AMI less than 0.001 and nm less than 0.005, in order to prevent an inner filter effect. The decrease of the tryptophan fluorescence emission was due to an energy transfer process (30) as shown by the shape of the emission spectrum; the shape was only reduced in magnitude, but no shift was observed. Furthermore, the decrease in tryptophan fluorescence emission was around 30% and could not be due to an inner filter effect under our conditions. There is an overlap between the absorption spectrum of quinacrine and the emission spectrum of tryptophan, a condition for resonance energy transfer if the two chromophores are in close proximity (30, 31).

Measurements of Action Potential-A freshly dissected bullfrog sciatic nerve (H. catesbiana) was desheathed with the aid of an operating microscope and refreshed in buffer (frog Ringers' solution, NaC1, 115 mM; KC1, 2.0 mM; CaCI?, 1.8 mM; NalHPO4, 1.3 mM; NaH2POI.H20, 0.7 mM, pH 7.0, 23 "C). The nerve was then laid across a series of electrodes embedded in a Lucite chamber in which the proximal end of the nerve was stimulated electrically and surface potentials could be recorded from the distal end. A segment of the nerve between the stimulating and recording electrodes was bathed in a plastic cup containing buffer or appropriate concentrations of a local anesthetic or drug. The nerve was stimulated during the duration of the bathing with DC square wave pulses of 0.1-0.2 ms duration, delay of 1.0 ms, frequency of 1 Hz, at a fixed or constant applied voltage (Grass SD9 stimulator). Applied voltages were in the low range, 150-300 mV, so that only the action potential of the Act nerve fibers was recorded. We used a Tektronix 5103N storage oscilloscope with a 5A20N differential amplifier, a 5A23N amplifier to determine the magnitude of the applied voltage, and a 5312N dual time base.

Experimentally, the nerve was bathed in local anesthetics or drugs for 30 min a t 23 "C until equilibrium was attained (or until there was no further decrease in the amplitude of the action potential). Appro- priate controls with buffer were performed in parallel. The nerve preparation was stable for 3-6 h, during which time there was less than a 10% decrease in the action potential. Nerve blockade was reversible upon resoaking the uerve in buffer. All compounds tested were dissolved in frog Ringers' solution. From time to time certain nerves showed marked deterioration with time merely after the desheathing step. Results from these nerves were discounted.

The apparent voltage dependence of local anesthetics on nerve blockage was determined by measuring the fractional height of the action potential uersus the applied stimulating voltage at different concentrations of each local anesthetic. Experimentally, the nerve was bathed for 30 min in a local anesthetic, after which time a voltage was applied stepwise, and the increase in amplitudes of the Aa action potential was recorded. This was repeated at two other concentrations for each local anesthetic. A plot of the percentage of reduction in amplitude versus concentration of local anesthetic for each applied voltage generated a series of curves from which the CW, (sciatic nerve) or concentration which reduces the original amplitude to 50% could be determined. The appropriate control of bathing with just buffer for 30 min and then applying a stimulating voltage was also done.

RESULTS

Fluorescence Emission of Bound Quinacrine-The fluo- rescence emission spectra of quinacrine in the presence and absence of axonal membranes are shown in Fig. 1A. The addition of saturating concentration of axonal membranes causes a 20 to 25% enhancement of the fluorescence intensity of quinacrine. Also, the fluorescence spectrum of bound quin- acrine (b) exhibits a 6- to 8-nm blue shift compared to quin- acrine in aqueous solution (a) , which has an emission maxi- mum at 500 nm (see also Ref. 29). This shift to a shorter wavelength, as well as the increase in the quantum yield, presumably reflects a lower polarity or higher viscosity of the fluorophore environment and suggests that quinacrine binds

8966 Quinacrine Binding to Mammalian Axonal Membranes

I I I I A

IpKa7.7) I E 4- .- b E 3 Ai

h

-si 2 - 3-

C

x .- + E

t

I-!

1 - b L

0 - I 400 450 500 5 5 0 600

Wavelength, nm

FIG. 1. Quinacrine binding to axonal membranes: spectral properties and Scatchard analysis. A, emission spectra of quina- crine. The spectra recorded are: a, 1 p~ quinacrine plus 11.4 pg/ml of axonal membrane protein, and 6, 1 p~ quinacrine. Excitation was at 350 nm. Details are given under “Experimental Procedures.” The emission maximum are, respectively, 500 nm and 492 nm for free and

to a nonpolar region in the membrane (30-32). Moreover, whether bound quinacrine is accessible to an external pertur- bant was answered by comparing the solvent quenching of quinacrine fluorescence in D20 uersus H20 (33, 34). The fluorescence of free quinacrine in 90% D20 is enhanced about 1.5-fold compared to HzO. Bound quinacrine is also enhanced to the same degree, about 1.5-fold, in 90% D20 compared to H20, indicating that the bound quinacrine molecules are accessible to solvent water (33,34).

The fluorescence enhancement observed upon binding to axonal membranes appeared to be saturable with quinacrine concentration in the range 0.1 to 8.0 PM. The binding param- eters KO and B,,, (number of sites) could be evaluated by Scatchard analysis (35), and a Scatchard plot was derived from two independent measurements. First, membrane pro- tein was added to a fixed concentration of quinacrine, called [Qlstandard, and a double reciprocal plot of protein concentra- tion versus fluorescence enhancement (AF) allowed extrapo- lation to the maximum fluorescence enhancement, called AF,,,,,, when all quinacrine was bound. Second, quinacrine in the range of 0.1 to 8.0 WM was added to a fixed concentration of membrane protein, and the fraction bound was calculated at each quinacrine concentration by the equation [Q]bound = [Qlsta,,dard X AF/AF,,,, where AF is the fluorescence enhance- ment for a given quinacrine concentration at [@. A Scatchard plot of quinacrine binding to axonal membranes is linear, indicating the existence of a single homogeneous population of binding sites (Fig. IB). In this case, the high affinity site has a KD of 0.6 p~ and Bmx in 10 nmol/mg of protein. DiEferent axonal membrane preparations showed a variation in Ku from 0.6 to 1.2 WM and B,,, from 2 to 4 nmol/mg of protein, although there was little variation within the same preparation.

Fluorescence binding data obtained from the energy trans- fer technique was also used to evaluate quinacrine binding (see ‘‘Fluorescence Measurements,” Set B), and both methods gave identical results. Nearly 30% of tryptophan emission was reduced upon quinacrine binding, indicating that membrane

bound quinacrine. The inset shows the molecular structure of quina- crine. B, Scatchard plot of quinacrine binding to axonal membranes. Quinacrine concentrations were in the range of 0.1 to 8.0 p ~ . The protein concentration was 8.3 pg/ml. In this case, the KU was 0.6 ,UM, and Bmax was 10 nmol/mg of protein. B,,, was found to range between 2 nmol/mg of protein and 4 nmol/mg of protein in most preparations.

TABLE I Effects of enzymes on quinacrine bound to axonal membranes Axonal membranes at 70 pg/ml of protein concentration were

preincubated for 10 min with 5 p~ quinacrine at 23 “C. Then, the fluorescence enhancement ( A F ) due to bound quinacrine was initially recorded and followed kinetically upon adding enzyme at 7.0 pg/ml of protein concentration (LKB 2210 1-channel recorder). After 30 to 60 min of incubation any change in the fluorescence enhancement was expressed as a fraction of the initial fluorescence enhancement. The addition of enzymes with comparable specific activities was checked.

Enzyme used Fraction of bound quinacrine remaining after treatment

Trypsin 0.05-0.10 a-Chymotrypsin 0.50-0.60 Pronase 0.70-0.75 Phospholipase A2 0.01-0.05 Phospholipase C 0.95-0.97 Phospholipase D 0.98-0.99 Ribonuclease A 0.97-0.99 Deoxyribonuclease I 0.98-0.99

proteins are implicated in this binding. Nature of the Quinacrine Binding Site in Axonal Mem-

branes-The effects of enzymes on quinacrine bound to ax- onal membranes are presented in Table I. Quinacrine binding is highly sensitive to protease treatment, especially so to trypsin. Axonal membranes (70 pg/ml) pre-equilibrated with 5 ~ L M quinacrine were treated with trypsin (7.0 ,ug/ml), and bound quinacrine was nearly completely eliminated (99-95%) within 30 min at 23 “C. Under similar conditions the other proteases a-chymotrypsin and pronase were less effective. On the other hand quinacrine binding was also sensitive to phos- pholipase A2 treatment (7.0 pg/ml), and bound quinacrine was completely abolished (by 95-99%) within 30 min at 23 “C. Interestingly, phospholipase C and phospholipase D had no effect upon bound quinacrine even when the ratio of enzyme to axonal membrane protein was raised from 1:lO to 1:1, ruling out a direct interaction of quinacrine with the phospholipid head groups in the membrane. Similarly, as a control, ribo-

Quinacrine Binding to Mammalian Axonal Membranes 8967

4 A

[Quinacrine1 i n ,uM

FIG. 2. Quinacrine binding to axonal membranes versus lipid extracts: cooperativity in the former. Fluorescence en- hancement (AF‘) of quinacrine upon binding to axonal membranes (0) versus lipid extract from axonal membranes (0). Lipid extraction was by the chloroform/methanol method of Folch et al. (36). Lipo- somes of the lipid extract were prepared in a cylindrical bath sonicator (Hicksville, NY). In this binding curve both the axonal membranes and liposomal extract were adjusted to the same phospholipid con- tent, 55 nmol of phospholipid, as determined by a phosphate assay (37, 38). The protein concentration in the axonal membranes was 50

8 A

-4 I I I I 2 N t m W

CPuin-inrl in pM

FIG. 3. Quinacrine binding is membrane- and tissue-specific. Fluorescence enhancement ( A F ) of quinacrine upon binding to axonal membranes (0) versus myelin membranes (0). The protein concen- tration in both membranes was 25 pg/ml as determined by either fluorescamine or Bio-Rad assays. Excitation was at 350 nm, and emission was at 500 nm. Bars indicate noise levels of measurements.

nuclease A and deoxyribonuclease I had no effect on quina- crine bound to axonal membranes (also tested at a ratio of l:l), ruling out DNA or RNA as possible binding sites. These results indicate that while a change in the state of the lipid in the membrane may influence the quinacrine binding the bind- ing site is likely to be in the protein components of the membrane.

Furthermore, a lipid extract prepared from axonal mem- branes (by the classical method of Folch et aL, Ref. 36) does not contain the high affinity quinacrine binding site, as shown clearly in Fig. 2 A . Here, the binding of quinacrine to both axonal membranes and a lipid extract was compared in sam- ples at identical phospholipid contents. From the figure it is c!ear that the binding of quinacrine to axonal membranes is saturable with a KO around 1.2 p ~ . The absence of this high

I B

[Ouinacrind in ,uM

pg/ml. Excitation was at 350 nm, and emission was at 500 nm. Bars indicate noise levels of measurements. B, Hill plot of quinacrine binding to axonal membranes. Data taken from the binding curve presented in A. The fluorescence enhancement ( A F ) ranged from 0.50 to 6.35 in arbitrary units, and the maximum fluorescence enhance- ment (AF,,,) was 6.50 units. The quinacrine concentration ranged from 0.25 to 8.0 p ~ . The axonal membrane protein concentration was 50 pg/ml. The straight line was fit by least squares analysis from a computer program and has a slope of 1.95.

-\ I ‘p Lo

l o g [Ouinacriml

B, fluorescence enhancement of quinacrine upon binding to corpus callosum (O), cerebral cortex (O), liver (O), and lung (W). The protein concentration in all membranes was 150 pg/ml. See “Experimental Procedures” for details of membrane preparation. Excitation was at 350 nm, and emission was at 500 nm.

affinity site in the lipid extract was also confirmed when testing a sample with a 3-fold higher phospholipid content than that given in Fig. 2 A . A low affinity site could not be accurately probed since quinacrine fluorescence is no longer linear with concentrations greater than 20 p ~ . Nevertheless, this result further suggests that the high affinity site in axonal membranes is either contained in the protein components of the membrane or it requires the integrity and intactness of the membrane structure itself for quinacrine binding.

Treatment of axonal membranes with high salt (2.0 M NaCl) and high pH (pH 11 with NaOH) results in a slight change of the protein profile as judged by sodium dodecyl sulfate-poly- acrylamide gel electrophoresis of the membranes after pellet- ing (data not shown). The membrane pellet obtained from such a treatment still contained the high affinity quinacrine

8968 Quinacrine Binding to Mammalian Axonal Membranes

binding site, KD = 1.2 p ~ , and the fluorescence enhancement (AH due to bound quinacrine was of the same magnitude as an untreated membrane pellet. Since this treatment has been known to remove selectively the peripherally bound proteins in biological membranes (39), it is reasonable to conclude that the quinacrine binding site is an integral membrane protein. Also, the proteins of the axonal membrane fraction appear to be mainly integral ones.

Finally, the cooperative nature of quinacrine binding can be seen in a Hill plot (Fig. 2B). A Hill coefficient of 1.95 was obtained, suggesting positive cooperativity in quinacrine bind- ing (40). Hill coefficients between 1.9 and 2.1 were found with other membrane preparations.

Membrane Specificity a n d Tissue Distribution of Quina- crine Binding Sites-Binding of quinacrine to the myelin membrane and axonal membrane fractions was compared at similar protein concentrations (Fig. 3A). Here, the high affin- ity quinacrine binding site was absent in myelin membranes at a protein concentration of 25 p g / d and 75 pg/ml. Thus, the high affinity quinacrine binding site is specific for the axonal membrane type and is enriched in axonal membranes versus myelin membranes during their preparation from my- elinated axons.

We also tested the extent of quinacrine binding in mem- branes prepared from nervous tissue and non-nervous tissue. It appears that the high affinity quinacrine binding site is present preferentially in neuronal membranes uersus non- neuronal membranes (Fig. 3B). From the figure it is clear that the brain regions corpus callosum and cerebral cortex contain the high affinity site (& = 1.0 p ~ ) , while liver and lung do not a t a membrane protein concentration of 150 pg/ml. Other brain regions that were tested similarly and found to contain the quinacrine binding site were cerebellum, spinal cord (cer- vical, lumbar, and thoracic regions), corpus striatum (caudate and putamen), thalamus, superior and inferior colliculus, pons, and medulla. The magnitude of the fluorescence enhancement

(An was largest for binding to corpus callosum than in any other brain region mentioned above (data not shown). Coin- cidentally, the corpus callosal region is richer in white matter or axons than any other brain region. Other tissues that lacked the quinacrine binding site were heart, pancreas, kidney, small intestine, and large intestine, all tested at a membrane protein concentration of 150pg/ml (data not shown). Protein concen- trations greater than 150 pg/ml were prohibitive because of the increased turbidity.

Competition with Local Anesthetics a n d Other Drug Classes-Quinacrine bound to axonal membranes is displaced effectively by several clinically useful local anesthetics (Fig. 4A). The effectiveness of quinacrine displacement (1Cmj0.*) cor- relates closely with the nerve blocking potency (Cs~r,) of local anesthetics tested on desheathed sciatic nerves, as shown clearly in Fig. 4B. Interestingly, quinacrine proved to be as potent a blocker of axonal conduction as dibucaine. A quina- crine concentration of 10 p~ reduced the amplitude of the Aa action potential of sciatic nerve by nearly 50%.

A series of drugs classified as tranquilizers/neuroleptics (41) was also tested for effectiveness in displacing bound quina- crine and blocking sciatic nerve conduction (Fig. 5), and a correlation line could be drawn although perceptibly not as close as with the local anesthetics. Incidentally, the opiates (naloxone, etorphine), benzodiazepines (chlordiazepoxide, di- azepam), and adrenergics (phenylephrine, prazosin, isoproter- enol, propanolol) were very weak displacers of bound quina- crine with ICsov values greater than 5 to 10 mM (data not shown). The barbiturates (general anesthetics) also displace bound quinacrine although they are much less effective than local anesthetics (Table 11).

A series of drugs that block excitable ionic channels was also studied. Tetraethylammonium and 3,4-diaminopyridine are drugs believed to specifically block potassium channels in nerve (2, 42). As shown in Table I1 3,4-diaminopyridine is unexpectedly 40-fold more potent than tetraethylammonium

-2 I I I I

L"""" 1.""" 0-7 -6 -5 -4 -3 -2

log (Ancsthetlc)

FIG. 4. Competition of local anesthetics for quinacrine bind- ing correlates with their nerve-blocking potencies. A, dose response curve of local anesthetic displacement of bound quinacrine. A plot of the fluorescence enhancement (AF) due to bound quinacrine versus the concentration of local anesthetic. Curves 1 through 6 are, respectively, for dibucaine, dimethisoquin, tetracaine, etidocaine, lid- ocaine, and procaine. The concentration of quinacrine was 5 p ~ , and the axonal membrane protein concentration was 125pg/ml . Excitation was a t 350 nm and emission was at 500 nm. None of the local anesthetics or drugs have intrinsic fluorescence a t these wavelength

log (IC 50%1 'conditions. B, correlation between the effectiveness of displacing bound quinacrine and the potency of sciatic nerve blockade. A plot is shown of the concentration of local anesthetic that reduces bound quinacrine by 50% (called ICrsc4,) versus the concentration of local anesthetic that reduces the amplitude of the Aa action potential in sciatic nerve by 508 (called CSO,). Details on the electrophysioby set-up and measurements are under "Experimental Procedures." Points 1 through 9 are, respectively, for quinacrine, dibucaine, tetra- caine, dimethisoquin, etidocaine, bupivicaine, prilocaine, lidocainz, and procaine.

Quinacrine Binding to Mammalian Axonal Membranes 8969

6

0

FIG. 5. Correlation as in Fig. 4, with data from tranquilizers and neuroleptics. Points 1 through 9, are, respectively, for a-flupen- thixol, thioridazine, haloperidol, chlorpromazine, P-flupenthlxol, flu- phenazine, imipramine, promethazine, and mesoridazine.

TABLE I1 Displacement of bound quinacrine by drugs

The concentration of quinacrine was 5 p~ and the axonal mem- brane protein concentration was 125 pg/ml. Excitation was at 350 nm, and emission was at 500 nm.

Drug IC;o.," r n M

Barbiturates Diphenylhy~anto~ 0.5 Pentobarbital 3.0 Secobarbital 4.6 Phenobarbital 5.7 Barbital 30

3,4-Diaminopyridine 0.43 Tetraethylammonium 18.0

Obtained from dose-response curves constructed as in Fig. a4.

Kt channel blockers

-3}-O 0 0 " *

-4 1 -s -

-6 -

Stimulating Voltage, mV

FIG. 6. Apparent dependence on stimulating voltage of- sciatic nerve blockade by quinacrine (0), dibucaine (@), and procaine ((3). A plot is shown of the concentration of local anesxhetic that reduced the amplitude of the action potential by 50% uersus the applied voltage. The frequency of the applied voltage was 10 Hz.

in displacing bound quinacrine, although both drugs are less potent than the local anesthetics. Tetrodotoxin is known to block the sodium channel in nerve (2, 43). Tetrodotoxin at 0.03 to 30 (LM had no effect upon quinacrine binding.

Apparent Voltage Dependence of Nerve BZockade by Quin- acrine-Surprisingly, we have found that the nerve blocking potency of quinacrine was highly dependent on the stimulat- ing voltage of the sciatic nerve (Fig. 6). Since a single nerve fiber fires in an all-or-none manner and generates an action potential of fixed amplitude, the maximum amplitude of the whole sciatic nerve is thus composed of the summation of all these individual nerve fibers Firing away (44). Thus, as an external voltage is applied with increasing magnitude to a sciatic nerve it displays a range from minimum to maximum amplitude of the action potential, which corresponds to re- cruiting more and more nerve fibers to fire. We found that at low voltages quinacrine was much more potent than dibucaine in nerve blockade. Neit,her of the two local anesthetics tested, namely procaine and dibucaine, exhibits as pronounced effect as that seen with quinacrine (Fig. 6).

DISCUSSION

Our results on quinacrine binding obtained under conditions Set A and Set B are in exact agreement. They suggest the existence in axonal membranes of a single class of high affinity sites (Fig. 1B). As energy transfer is a short range process, our measurements indicate a close proximity of binding t,o certain tryptophan residues (31). The increase in quantum yield and blue shift in emission maximum of 6 to 7 nm is consistent with the notion that bound quinacrine senses an environment less polar or more viscous than aqueous quinacrine (30-34). More- over, bound quinacrine appeared to be rather freely accessible to solvent water as shown in our comparison experiment with D20 (33, 34). Such results (less polar, more viscous environ- ment, and solvent accessibility) are consistent with the re- ported properties of membrane-bound emphiphiles (30-34). Since the molecule quinacrine has a strongly basic diethyl- amino group with a pK, near 10.4 (see Fig. 1A, inset), a t pH 7.4 (physiological pH) this group will be protonated and the predominant species (99.9%) will be positive charged. Thus, quinacrine fulfills the criteria for an amphiphile (34).

The high affinity site appears to be an integral membrane protein. The site is detected by energy transfer measurements, destroyed by proteolytic enzymes (Table I), lost upon chlo- roform/methanol extraction and absent in a lipid ti-action prepared therefrom (Fig. 2 A ) , and retained in a membrane pellet previously treated with high salt (2.0 M NaCl) and high pH (pH 11 with NaOH), a treatment which removes selec- tively the peripherally bound proteins (39). The marked sen- sitivity of the site to phospholipase AS treatment (Table I) is consistent with our assertion that the integrity or intactness of the membrane structure is essential for binding (45). This sensitivity could be explained by either the loss of membrane phospholipids specific for binding or detergent solubilization of the membrane by endogenously released lysophosphatides.

The exact nature of the high affinity site cannot be specified. At least two models can be conceived: one, it is a protein; two, it is at the level of interaction of some protein and its phos- pholipid environment (perhaps annular lipid) (3). However, any model formulated should take into account the coopera- tive nature of quinacrine binding (Fig. 2B) and the lack of effect of phospholipases C and D on binding (Table I), the latter result ruling out the direct interaction of quinacrine with phospholipid head groups.

A low affinity site could not be accurately probed since quinacrine fluorescence is no longer linear with concentration about 20 (LM (29). However, we do not rule out a nonspecific

8970 Quinacrine Binding to Mammalian Axonal Membranes

binding site on membrane lipids as a secondary mechanism for the action of local anesthetics. As shown clearly in Fig. 4A not all the bound quinacrine is displaced even at high concen- trations of local anesthetics. Quinacrine is known to bind to purine nucleotide triphosphates and DNA (46). Since our preparation is devoid of nuclei and free purines (23) and ribonuclease A and deoxyribonuclease I had no effect upon bound quinacrine (Table I), we believe that nucleotide binding is unrelated to the phenomena described here.

The tissue distribution of quinacrine binding reveals a clear preference for neuronal tissue. The high affinity site (KO of 0.6 to 1.2 pM) is present in neuronal membranes but apparently absent from non-neuronal membranes (Fig. 3B). Moreover, the brain region which contains the highest percentage of axons or white matter, the corpus callosum, also exhibits the largest magnitude in fluorescence enhancement. The high affinity site is also enriched in axonal membranes over myelin membranes during their preparation from the myelinated axon source, the corpus callosum (Fig. 3A). Upon comparing the magnitude of the fluorescence enhancement of corpus callosal membranes at 150 pg/ml (Fig. 3B) with that of axonal membranes at 25 pg/ml (Fig. 3A), it is apparent that the high affinity site has become highly enriched in axonal membranes during the preparative steps.

Since protein is clearly involved in the high affinity quina- crine binding site, the question arises as to its identity. The close correlation between local anesthetic displacement of bound quinacrine and potency of nerve blockade suggests the existence of a local anesthetic receptor site in axonal mem- branes (Fig. 4B). The pharmacology of this receptor site seems, at present, unclear. A less close correlation can be drawn for the class of drugs called tranquilizers/neuroleptics (Fig. 5), which have also been shown to be potent blockers of axonal conduction (41). The opiates, adrenergics, and benzo- diazepines do not interact with this receptor site (data not shown).

We tested drugs which bind specifically to excitable ionic channels to determine if they have any effect upon quinacrine binding. Tetraethylammonium and 3,4-diaminopyridine, drugs which block the potassium channel in nerve (42), clearly have a differential effect upon bound quinacrine (Table 11). Tetrodotoxin, a toxin believed to block the sodium channel pore (43), had no effect upon quinacrine binding. The lack of interaction between tetrodotoxin and local anesthetics in nerve has been reported before (47). It might not be surprising if other regulatory sites on the excitable sodium/potassium channels (2, 19, 43) or novel receptor proteins could be in- volved in quinacrine binding.’ In fact, two recent lines of evidence suggest the very existence of multiple local anes- thetic receptor sites. First, Khodorov et al. (48) found via voltage clamp studies of frog sciatic nerve the existence of several types of receptor sites through which local anesthetics might exert their blocking action of sodium permeability. A receptor site designated R1 seemed to inactivate rapidly with an uncharged molecule of tertiary amino-local anesthetics or neutral benzocaine, while a second receptor RP was responsible for slow inactivation with tertiary amines of unknown charge applied to the outer side of the nerve membrane. The recep- tors designated R3 and Rg were responsible for the use-de- pendent inhibition by local anesthetics (2) and interacted with permanently charged quaternary or protonated tertiary amine anesthetics applied to the inner side of the nerve membrane.

‘ We have not yet fully tested batrachotoxin (43), veratridine, and histrionicotoxin (19) on quinacrine binding because of their solubility requirements in organic solvents, e.g. ethanol, the latter being a potent displacer of bound quinacrine. Currently we are studying other water-soluble organic solvents.

Second, NMR data on binding deuterated local anesthetics to artificial membrane vesicles also suggest multiple binding sites, with the binding site for the charged and uncharged species being different, the former probably being closer to the membrane-water interface while the latter deeply embed- ded within the membrane (49). Thus, definitive identification of the local anesthetic receptor protein must await solubili- zation and purification of the receptor, which we are currently pursuing by affinity chromatography.

Finally, the “apparent” dependence on applied voltage (Fig. 6) can be accounted for by at least three possibilities: 1) quinacrine is intrinsically more potent than either dibucaine or procaine, in that at low voltages where only a few nerve fibers are f i n g (the larger ones, 10 to 20 p in diameter) quinacrine is potent enough to block them; 2) quinacrine is preferentially selective for certain Aa nerve fibres, perhaps motor versus sensory, at low applied voltages; 3) quinacrine is a voltage- and frequency-dependent drug, putatively like other local anesthetics ( 2 ) , and is a selective blocker of the open conformation uersus the closed conformation of the excitable sodium channel as first demonstrated by Adams and Feltz (15). This third possibility should be seriously entertained in light of a recent paper demonstrating the inhibition of the inactivation gate in sodium channels in a voltage-sensitive manner by sea anemone toxin, ATX-II(50). Local anesthetics have also been shown to inhibit the inactivation gate in sodium channels (2). In passing it should be mentioned that quinacrine might be of use clinically as lidocaine or pro- cainamide have been in treating cardiac arrhythmias.

The number of quinacrine binding sites reported here, B,,,., = 2 to 4 nmol/mg, suggests that the local anesthetic receptor site is a major constituent of axonal membranes. Theoreti- c d y , a putative 50,000-dalton receptor protein with a 1:1 molar ratio of bound quinacrine to receptor would comprise 10-20% of the axonal membrane proteins. Such an enriched receptor protein could easily be identified on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of axonal membrane proteins; however, mere visual inspection of a representative gel reveals seuerczl major protein bands which could be implicated as the receptor species (data not shown). Interestingly, affinity chromatography eluates of detergent- solubilized axonal membranes do reveal by gel electrophoresis a small protein, about 20,000 daltons, which clearly comigrates with the starting membrane proteins. Definitive characteri- zation of the local anesthetic receptor by affinity chromatog- raphy is currently underway in our laboratory.

Acknowledgments-We would like to give special thanks to Dr. Solomon H. Snyder for invaluable suggestions and advices and to Dr. George H. DeVries for allowing us to read his manuscript before publication and guidance in making axolemmal preparations. Thanks to Dean Chang, Dr. Robert DeVoe, Jonathan Freedman, Barry Knox, Jose Pardo, Dr. Engin Serpersu, and Dr. Justin Teissie for helpful discussions and technical assistance.

REFERENCES 1. Richards, C. D. (1978) in Biochemistry of Cell Walls and Mem-

branes 11, (Metcalfe, J. C., ed) Vol. 19, pp. 157-220, University Park Press, Baltimore

2. Strichartz, G. (1976) Anesthesiology 45, 421-441 3. Lee, A. G. (1976) Nature (Lond.) 262,545-548 4. Pringle, M. J., and Miller, K. W. (1978) Biochem. Biophys. Res.

5. Lee, A. G. (1976) Biochim. Biophys. Acta 448,34-44 6. Ueda, I., Tashiro, C., and Arakawa, K. (1977) Anesthesiology 46,

7. Tsong, T. Y., Greenberg, M., and Kanehisa, M. I. (1977) Biochem-

8. Mountcastle, D. B., Biltonen, R. L., and Halsey, M. J. (1978)

Commun. 85, 1192-1198

327-332

istry 16,3115-3121

Proc. Natl. Acad. Sci. U. S. A. 75,4906-4910

Quinacrine Binding to Mammalian Axonal Membranes 8971

9. Narahasi, T., and Frazier, D. T. (1975) J. Pharmacol. Exp. Ther.

10. Narahashi, T., Frazier, D. T., and Yamade, M. (1970) J . Phar-

11. Hille, B. (1977) J. Gen. Physiol. 69,497-515 12. Ruff, R. L. (1976) Biophys. J. 16,433-439 13. Ruff, R. L. (1977) J. Physiol. (Lond.) 264,89-I24 14. Adams, P. R. (1977) J. Physiol. (Lond.) 268,291-319 15. Adams, P. R., and Feltz., A. (1977) Nature (Lond.) 269,609-611 16. Steinbach, J. H. (1977) Biophys. J. 18,357-358 17. Grunhagen, H., and Changeux, J. P. (1976) J. Mol. Biol. 106,

18. Grunhagen, H., Iwatsubo, M., and Changeux, J. P. (1977) Eur. J. Biochem. 80,225-242

19. Heidmann, T., and Changeux, J . P. (1978) Annu. Rev. Biochem. 47,317-357

20. Sobel, A., Heidmann, T., Hofler, J., and Changeux, J . P. (1978) Proc. Natt. Acad. Sei. U. S. A. 75, 510-514

21. Eldefrawi, M. E., Eldefrawi, A. T., Mansour, N. A., Daly, J. W.,

5474-5484 Witkop, B., and ~buquerque , E. X. (1978) Biochemistry 17,

22. Tsai, M.-C., Oliveira, A. C., Albuquerque, E. X., Eldefrawi, M. E., and Eldefrawi, A. T. (1979) Mol. Pharmacol. 16, 382-392

23. DeVries, G. H. (1981) in Research Methods in Neurochemistry (Marks, N., and Rodnight, R., eds) Vol. 5, pp. 338-, Plenum Press, New York

24. Zetusky, W. J., Cdabrese, V. P., Zetusky, A. L., Anderson, M. G., Cullen, M., and DeVries, G. H. (1979) J. Neurochem. 32,

25. DeVries, G. H., Matthieu, J. M., Beny, M., Chicheportiche, R., Lazdunski, M., and Dolivo, M. (1978) Brain Res. 147, 339-352

26. Sweadner, K. J. (1979) J. Biol. Chem. 254,6060-6067 27. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber,

194,506-513

macol. Exp. Ther. 171, 32-44

497-516

1103-1109

W., and Weigle, M. (1972) Science 178,871-873

28. DeVries, G. H., Payne, W., and Saul, R. G. (1981) Neurochem. Res. 6,521-537

29. Massari. S.. Dell'Antone. P., Colonna. R.. and Azzone, G. F. (1974) , . Biochem&y 13, 1038-1043

cisco 30. Freifelder, D. (1976) Physical Biochemistry, Freeman, San Fran-

31. Stryer, L. (1968) Science 162,526-533 32. Angelides, K. J. (1981) Biochemzs~ry 20,4107-4118 33. Radda, G. K. (1971) Biochem. J. 122,385-396 34. Radda, G. K. (1975) Methods Membr. Biol. 4,97-188 35. Scatchard, G. (1949) Ann. N. Y. Acad. Sei. 51,660-672 36. Folch, J., Lees, M., and Sloane Stanley, G. H. (1957) J. Biol.

37. Bartlett, G. R. (1959) J. BioZ. Chem. 234, 466-468 38. Chen, P. S., Jr., Toribara, T. Y., and Warner, H. (1956) Anal.

39. Steck, T. L., and Yu, J. (1973) J. Supramol. Struct. 1,220-232 40. Dahlquist, F. W. (1978) Methods Enzymol. 48, 270-299 41. Seeman, P., Staiman, A., and Chau-Wong, M. (1974) J. Phar-

42. Kirsch, G. E., and Narahashi, T. (1978) Biophys. J. 22, 507-512 43. Catterall, W. A. (1980) Annu. Rev. Pharrnacol. Toxicol. 20,14-43 44. Covino, B. G., and Vassallo, H. G. (1976) Locat Anesthetics,

45. Reed, J. K. (1981) Biochim. Biophys. Acta 646,43-50 46. Krey, A. K., and Hahn, F. E. (1974) Mol. Pharmacol. 10,686-695 47. Benzer, T. I., and Raftery, M. A. (1972) Proc. Natl. Acad. Sei. U.

48. Khodorov, B., Shishkova, L., Peganov, E., and Revenko, S. (1976)

49. Boulanger, Y., Schreier, S., Leitch, L. C., and Smith, I. C. P.

50. Miyake, M., and Shibata, S. (1981) Mol. Pharrnacol. 20,453-456

Chem. 226,497-509

Chem. 28,1756-1758

macol. Exp. Ther. 190, 123-130

Grune and Stratton, New York

S. A. 69,3634-3637

Biochim. Biophys. Acta 433,409-435

(1980) Can. J. Biochem. 58, 986-995