reserpine: interactions with batrachotoxin and brevetoxin sites on voltage-dependent sodium channels

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Cellular and Molecular Neurobiology [cemn] pp454-cemn-371696 April 22, 2002 14:25 Style file version Oct 23, 2000

Cellular and Molecular Neurobiology, Vol. 22, No. 1, February 2002 ( C© 2002)

Reserpine: Interactions With Batrachotoxin and BrevetoxinSites on Voltage-Dependent Sodium Channels

Andrew Flowers,1,2 Kenolisa Onwueme,1 Cyrus R. Creveling,1 and John W. Daly1,3

Received October 15, 2001; accepted October 20, 2001

SUMMARY

Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synap-toneurosomes with an IC50 of about 1 µM. In the presence of brevetoxin the IC50 in-creased to about 80 µM. Reserpine inhibited binding of batrachotoxinin-A [3H]benzoate([3H]BTX-B) binding in a complex manner causing a partial inhibition from 0.001 to0.08 µM, then a rebound stimulation from 0.1 to 0.8 µM, followed by complete inhibi-tion by 80 µM. The stimulation was prevented by the presence of brevetoxin; reserpinethen smoothly inhibited binding with an IC50 of about 1 µM. Reserpine at 1 µM slightly re-duced the off-rate of [3H]BTX-B binding measured in the presence of veratridine, while ata concentration of 50 µM it enhanced the off-rate, presumably by an allosteric mechanism.Reserpine at 0.3–10 µM elicited a partial inhibition of the binding of [3H]brevetoxin-3.The local anesthetic dibucaine had effects similar to reserpine: It partially inhibited bind-ing of [3H]brevetoxin. The presence of brevetoxin reduced the potency of dibucaine as aninhibitor of batrachotoxin-elicited sodium influx from an IC50 of about 2 µM to an IC50 ofabout 50µM. The results suggest that reserpine binds at both a local anesthetic site to causeallosteric inhibition of batrachotoxin-binding and action, but that it also binds to anothersite causing, like brevetoxin, an enhancement of batrachotoxin-binding and action. Localanesthetics also may bind to the brevetoxin site.

KEY WORDS: sodium channels; batrachotoxin; brevetoxin; reserpine.

INTRODUCTION

A variety of biologically active agents have, in addition to activity at specific tar-gets, the ability to block ion channels. For example, the indole alkaloid yohimbine,an α2-adrenergic antagonist, is well-known to have local anesthetic activity and,therefore, to block batrachotoxin-elicited sodium influx through voltage-dependentsodium channels (cf. McNeal et al., 1985; Zimanyi et al., 1988). Reserpine, a

1 Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health, Bethesda, Maryland.

2 Present address: Centre for Phytochemistry, Southern Cross University, Lismore, New South Wales 2480,Australia.

3 To whom correspondence should be addressed at Laboratory of Bioorganic Chemistry, National Instituteof Diabetes and Digestive and Kidney Diseases, Building 8, Room 1A17, National Institutes of Health,Bethesda, Maryland 20892; e-mail: [email protected].

1

0272-4340/02/0200-0001/0 C© 2002 Plenum Publishing Corporation

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2 Flowers, Onwueme, Creveling, and Daly

structurally related indole alkaloid, which is well known for depletion of biogenicamines, potently inhibits, like local anesthetics, the binding of [3H]BTX-B to voltage-dependent sodium channels in brain synaptosomes (McNeal et al., 1985). However,the affinity of reserpine for the amine-uptake system of storage vesicles (Schermanand Henry, 1984) is severalfold greater than the affinity of reserpine for sodiumchannels.

The effects of reserpine on binding of toxins to voltage-dependent sodium chan-nels and the blockade of such channels by reserpine have now been investigated indetail. Reserpine appears to have at least two modes of interaction with sodiumchannels, one of which appears because of interaction with the brevetoxin site, whilea second, lower affinity interaction appears because of binding at a local anestheticsite that allosterically inhibits binding of batrachotoxin. The results provide furtherevidence for the complex regulation of sodium channel function by toxins and drugs(for reviews see Catterall, 1992; Strichartz et al., 1987).

METHODS

Materials

Batrachotoxinin-A [3H]benzoate ([3H]BTX-B) was from NEN (Boston, MA).22NaCl was from Amersham (Arlington Heights, IL). [3H]Brevetoxin (PbTx-3) wasgenerously provided by Dr Daniel G. Baden (University of North Carolina atWilmington). Batrachotoxin was isolated as described (Tokuyama and Daly, 1983).Brevetoxin (PbTx-3) was from RBI (Natick, MA). Veratridine, tetrodotoxin, scor-pion venom (Leiurus quinquestriatus), dibucaine, and reserpine were from Sigma(St. Louis, MO). Other agents were from standard commercial sources.

Preparation of Synaptoneurosomes

Guinea pig synaptoneurosomes were prepared essentially as described(Hollingsworth et al., 1985). Briefly, ice-cold cerebral cortex from male Hartley guineapigs (200–250 g) was homogenized in 7–10 volumes of ice-cold HEPES incubationbuffer, consisting of 50 mM HEPES buffer, pH 7.4 containing 130 mM choline chlo-ride, 5.4 mM KCl, 0.8 mM MgS04, and 5.5 mM glucose, with 7/8 strokes in a glass–glassconical homogenizer. For sodium flux assays the HEPES incubation buffer contained1 mg/mL bovine serum albumin and the initial homogenate was first filtered throughtwo layers of 100 µM mesh nylon by using a Millipore filter holder, followed by fil-tration through a 10µM Millipore filter (LC WP047) and washing with another 7 mLof ice-cold buffer. The combined filtrate was then centrifuged at 1800g for 15 min,and resuspended in buffer to provide a synaptoneurosome preparation with about10 mg/mL protein for sodium flux assays.

Sodium Influx Assay

The sodium flux studies were conducted essentially as described (Gusovsky et al.,1987). Briefly, the guinea pig synaptoneurosome preparation was incubated in the

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Reserpine and Sodium Channels 3

HEPES incubation buffer containing 1 mg/mL bovine serum albumin and various testagents in a volume of 100µL for 10 min at 37◦C. The 22NaCl (1.3µCi/mL) was addedin a 150 µL volume of a HEPES influx buffer containing 50 mM HEPES (pH 7.4),2.66 mM NaCl, 128 mM choline chloride, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mMglucose, 1 mg/mL bovine serum albumin, and 5 mM ouabain. The final volume was250 µL. The influx buffer contained the same test agents as in the incubation buffer.Influx of 22Na+ was stopped after 10 s by adding 4 mL of an ice-cold HEPES washbuffer, followed by collection on 0.45 µM Gelman GM-6 filter and washing twicewith ice-cold HEPES wash buffer. The HEPES wash buffer contained 5 mM HEPES(pH 7.4), 163 mM choline chloride, 0.8 mM MgSO4, 1.8 mM CaCl2, and 1 mg/mLbovine serum albumin. The filters were dissolved in Filtron-X (National Diagnostics)for gamma-ray scintillation counting. The sodium-channel specific uptake of 22Na+

was determined by subtracting nonspecific uptake obtained in the presence of 5 µMtetrodotoxin from the total uptake.

Batrachotoxinin-A [3H]Benzoate Binding Assay

The binding assay with [3H]BTX-B was conducted essentially as described(Catterall et al., 1981). Briefly, the guinea pig synaptoneurosome preparation(∼200 µg protein) was added to HEPES incubation buffer containing 10 nM[3H]BTX-B (sp. act. 14 Ci/mmol), 1 µM tetrodotoxin, 0.03 mg scorpion venom, andvarious agents to give a final volume of 250 µL. Incubations were for 30 min at 37◦Cand were terminated by addition of 3 mL ice-cold HEPES wash buffer, followedby rapid filtration through Whatman GF/C filters by using a Brandel Cell Harvester(Brandel, Gaithersburg, MD). The filters were washed twice with 3 mL of ice-coldwash buffer. Filters were placed in scintillation vials with 4 mL Hydrofluor for liq-uid scintillation counting. Nonspecific binding of [3H]BTX-B was determined in thepresence of 100 µM veratridine.

In off-rate experiments, synaptoneurosomes were incubated with 10 nM[3H]BTX-B as described above for 90 min. After centrifugation and resuspension,aliquots were further incubated of 37◦C for 5, 10, 20, 40, and 60 min in the absenceof further additions, or with 300 µM veratridine to prevent rebinding of [3H]BTX-B,or with test agent alone or with test agent in the presence of 300 µM veratridine.Incubations were terminated by addition of 3 mL ice-cold HEPES buffer, followedby rapid filtration as described above.

[3H]Brevetoxin-3 Binding Assay

The binding assay with [3H]brevetoxin-3 (PbTx-3) was essentially described(Poli et al., 1986) except that the incubation was at 37◦C instead of 4◦C. Briefly, theguinea pig synaptoneurosome preparation (∼200µg protein) was added to microfugetubes containing 5 nM [3H]PbTx-3 (sp. act. 10–15 Ci/mmol) and various agents inthe HEPES incubation buffer with 2 mg/mL of bovine serum albumin to give a finalvolume of 250 µL. Incubations were for 30 min at 37◦C and were terminated bycentrifugation for 30 s at 15,000g. The supernatant was removed by aspiration, andthe pellet was suspended by vortexing in 0.5 mL of ice-cold HEPES wash buffer. Thetubes were centrifuged for 30 s at 15,000g and the supernatant aspirated. Another

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0.5 mL wash buffer was added followed by vortexing and transfer to scintillationvials with 4 mL Hydrofluor for liquid scintillation counting. Nonspecific binding wasdetermined in the presence of 10 µM brevetoxin.

RESULTS

Reserpine caused a dose-dependent inhibition of batrachotoxin-elicited sodiuminflux in synaptoneurosomes (Fig. 1(A)). The IC50 was about 1 µM in the absenceand 80 µM in the presence of brevetoxin. The local anesthetic dibucaine also causeda dose-dependent inhibition of batrachotoxin-elicited sodium with an IC50 of about1 µM in the absence and 50 µM in the presence of brevetoxin (Fig. 1(B)).

Reserpine had complex effects on the binding of [3H]BTX-B to synaptoneu-rosomes (Fig. 2(A)). At very low concentrations (<0.1 µM), there was a modestbut highly reproducible dose-dependent inhibition of binding by reserpine that max-imized at about 0.08 µM. There followed a rebound stimulation of binding fromabout 0.1 to 0.8µM. At higher concentrations a complete dose-dependent inhibitionthen occurred. The stimulation was abolished in the presence of brevetoxin, whilethe inhibition was little affected (Fig. 2(B)). Dibucaine caused only an inhibition ofbinding of [3H]BTX-B with an IC50 value of 1.3 µM (data not shown).

Reserpine, at a low concentration of 1 µM, slightly slowed the off-rate for[3H]BTX-B binding as measured in the presence of 300 µM veratridine (Fig. 3(A)).At a concentration of 25 µM reserpine alone enhanced the off-rate of [3H]BTX, buthad no significant effect on the off-rate in the presence of veratridine (Fig. 3(B)).At a concentration of 50 µM, reserpine alone enhanced the off-rate both in theabsence and to a slight extent in presence of veratridine (Fig. 3(C)). Brevetoxin at0.3 µM, like reserpine at 1 µM, reduced the off-rate in the presence of veratridine(Fig. 3(D)). The off-rate of [3H]BTX-B binding was accelerated from half-time of32 min in presence of veratridine alone to 19 min in the presence of veratridine anda 6 µM concentration of the local anesthetic dibucaine (data not shown). Dibucainealone, as expected of a local anesthetic (Postma and Catterall, 1984), enhanced theoff-rate resulting in a half-time of 26 min.

The binding of [3H]brevetoxin-3 to synaptoneurosomes at 37◦C was partiallyinhibited by reserpine with a maximal inhibition of about 40% (Fig. 4). The half max-imal inhibition (20%) occurred at about 0.3 µM reserpine. There was no inhibitionof [3H]brevetoxin-3 binding by reserpine at 4◦C (data not shown). Similarly, the localanesthetic dibucaine had no effect on [3H]brevetoxin-3 binding at 4◦C, but caused amaximal inhibition of about 40% at 37◦C with a half-maximal effect at about 0.5 µM(Fig. 4). Other local anesthetics, such as bupivacaine and piperocaine, at 10 µMcaused only a 20% inhibition of binding of [3H]PbTx-3, but at lower concentrationsappeared to cause slight stimulations of binding (data not shown). Brevetoxin fullyinhibited binding both at 4◦C and at 37◦C (data not shown and Fig. 4).

DISCUSSION

The interaction of toxins and drugs with voltage-sensitive sodium channels hasbeen studied extensively for many years (Catterall, 1992; Strichartz et al., 1987).

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Fig. 1. Inhibition of batrachotoxin-elicited influx of 22sodium ions by reserpine with guineapig synaptoneurosomes. (A) Effect of reserpine on influx elicited by 1µM batrachotoxin and0.3 mg scorpion venom in the absence (d) or presence (s) of 0.1 µM brevetoxin. (B) Effectof dibucaine on influx elicited by 1 µM batrachotoxin and 0.3 mg scorpion venom in theabsence (j) or presence (h) of 0.1 µM brevetoxin. Assays as described in Methods. Valuesare means± SEM (n = 3).

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Fig. 2. Inhibition of binding of [3H]BTX-B binding by reserpine with guinea pig synap-toneurosomes. (A) Inhibition by reserpine in the presence of 0.03 mg scorpion venom.(B) Inhibition by reserpine in the presence of 0.03 mg scorpion venom and 0.1 µM breve-toxin. Assays as described in Methods. Values are means± SEM (n = 3).

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Fig. 3. Dissociation of [3H]BTX-B from guinea pig synaptoneurosomes. Effect of reserpine.Synaptoneurosomes were equilibrated with [3H]BTX-B for 90 min. After resuspension theoff-rate was measured in the presence of 300 µM veratridine to prevent reassociation of the[3H]BTX-B with the binding site. Symbols: e No veratridine; h Veratridine; s Veratridineplus reserpine or brevetoxin; d Reserpine or brevetoxin alone. (A) Effect of 1 µM reserpine.(B) Effect of 25µM reserpine. (C) Effect of 50µM reserpine. (D) Effect of 0.3µM brevetoxin.The veratridine curve in (C) is the average of three experiments, where the SEM was from 0.02to 0.07. In other cases, representative experiments are shown.

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Fig. 3. (Continued )

Several toxin sites have been defined, including a site at which tetrodotoxin andsaxitoxin block the channel pore, a site within the channel at which batrachotoxinand certain other toxins stabilize an open form of the channel, a site at which α-scorpion toxins inhibit inactivation of the channel, a site at which β-scorpion toxinshifts the voltage-dependency of channel to favor opening, and a site at which

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Fig. 4. Inhibition of binding of [3H]brevetoxin-3 by reserpine and dibucaine with guinea pigsynaptoneurosomes. Inhibition by reserpine (h) by dibucaine (s) and by brevetoxin (m). Assaysat 37◦C as described in Methods. Values are means± SEM (n = 3) for reserpine and brevetoxinand are from a representative experiment for dibucaine. Standard errors were smaller than thesymbol in many instances.

brevetoxins shift the voltage-dependency of the channel so as to favor opening. Thereis a complex interplay between the effects of different toxins on the function of thechannels. In addition, many drugs affect the binding and action of the toxins throughallosteric effects, that are often, as is the case for local anesthetics. Local anestheticsinhibit the binding of a radioactive batrachotoxin analog [3H]BTX-B, (Creveling andDaly, 1992; Creveling et al., 1983; McNeal et al., 1985; Nishizawa et al., 1988; Postmaand Catterall, 1984; Willow and Catterall, 1982). Although the inhibition had ap-peared competitive, it proved to be due to an allosteric effect of the local anesthetic,which markedly increased the off-rate for dissociation of the [3H]BTX-B (Postmaand Catterall, 1984). However, certain compounds can both inhibit the on-rate andenhance the off-rate (cf. Sheldon et al., 1994). Recently, a close localization of batra-chotoxin and local anesthetic binding sites has been proposed (Linford et al., 1998).The toxins veratridine and aconitine are true competitive inhibitors of [3H]BTX-Bbinding and have been used as such to measure off-rates of the [3H]BTX-B. Thus, theoff-rate of [3H]BTX-B binding can be determined by the addition of a high concen-tration of veratridine, which as a true competitive inhibitor prevents any rebinding of[3H]BTX-B (Postma and Catterall, 1984). The binding of [3H]BTX-B is enhanced byα-scorpion toxins (Catterall et al., 1981) and by brevetoxins (Catterall and Gainer,1985; Sharkey et al., 1987; Trainer et al., 1993) through allosteric mechanisms. Localanesthetics also enhance the off-rate for [3H]BTX-B (Postma and Catteral, 1984).Brevetoxins also affect α-scorpion toxin binding (Cestele et al., 1995, 1996).

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Reserpine, like many drugs with local anesthetic activity, inhibits binding of[3H]BTX-B (McNeal et al., 1985), as well as batrachotoxin-elicited sodium influx(Fig. 1(A)). The present studies revealed that reserpine has complex effects on[3H]BTX-B binding, as illustrated in Fig. 2(A), where concentrations of reserpinefrom 0.1 to 0.8 µM caused a rebound stimulation of binding. Effects on off-ratesfor [3H]BTX-B binding reflected the complex, concentration-dependent effects ofreserpine, namely a decrease in off-rates at a low concentration (1 µM) of reserpine,an increase at high concentrations (50 µM) and no effect at an intermediate concen-tration (25 µM) (Fig. 3(A)–(C)). Presumably, the stimulatory and inhibitory effectsof reserpine on [3H]BTX-B binding off-set each other at the intermediate concen-tration. Brevetoxin at 0.1 µM reduced the off-rate for [3H]BTX-B in the presenceof veratridine (Fig. 3(D); cf. Sharkey et al., 1987), much like 1 µM reserpine.

In an attempt to elucidate the mechanism whereby low concentrations of reser-pine enhance binding of [3H]BTX-B, the effect of other agents known to enhancebinding of [3H]BTX-B were assessed. Pyrethroids did not prevent the reserpine-elicited enhancement of [3H]BTX-B binding (data not shown), but brevetoxin did(Fig. 2(B)). In the presence of brevetoxin, reserpine showed only a concentration-dependent inhibition of binding of [3H]BTX-B binding with an IC50 of about 1 µM(Fig. 2(B)). A much higher concentration of reserpine, namely about 80 µM, wasrequired to cause a 50% blockade of sodium influx stimulated by a combinationof 1 µM batrachotoxin, scorpion venom, and 0.1 µM brevetoxin (Fig. 1(B)). In theabsence of brevetoxin, reserpine inhibited batrachotoxin-elicited sodium influx withan IC50 of about 1 µM (Fig. 1(A)). There was an indication of a slight stimulatoryeffect of reserpine at 0.1µM. Reserpine appeared to interact with high affinity at thebrevetoxin site causing only a maximal 40% inhibition of [3H]brevetoxin-3, but withan IC50 of less than 1 µM.

The complex effects of reserpine are undoubtably mediated by more than onesite. The interaction of reserpine with the brevetoxin-site is novel and unanticipated.It now has been shown that local anesthetics, including dibucaine, also interact withthe brevetoxin site to cause inhibition of binding of [3H]brevetoxin (Fig. 2(C) anddata not shown). Such an interaction was not manifest at 4◦C, the temperature atwhich prior studies of brevetoxin binding were conducted (Poli et al., 1986). Ap-parent interactions of local anesthetics and brevetoxins were unanticipated and in-deed, based on data with chimeric sodium channels, it was concluded that the so-called local anesthetic site was not coupled to the brevetoxin-binding site (Linfordet al., 1998). This may be true since the effects of reserpine (and local anesthet-ics) on brevetoxin binding are most likely not mediated through the so-called lo-cal anesthetic site. The molecular nature of interactions of these compounds withthe brevetoxin site remains obscure, but appears temperature-dependent, i.e., notmanifest at 4◦C. The stimulation of [3H]BTX-B binding by reserpine is reminis-cent of the stimulation by brevetoxin and is eliminated in the presence of breve-toxin. Such data suggest that reserpine has agonist or partial agonist activity at thebrevetoxin site. In contrast, the local anesthetic dibucaine causes only inhibitionsuggesting that it does not have agonist activity at the brevetoxin site or that suchactivity is masked by a potent interaction of dibucaine at the local anesthetic site.The inhibition of batrachotoxin-elicited sodium influx and [3H]BTX-B binding by

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reserpine appears likely to involve the so-called local anesthetic site. The great reduc-tion in potency of reserpine (Fig. 1(A)) in blocking batrachotoxin-elicited sodiumflux in the presence of brevetoxin may only reflect a greater stabilization of theopen form of the channel by the presence of both toxins. There is a similar re-duction in potency of local anesthetics, including dibucaine, when sodium flux isstimulated by a combination of batrachotoxin and brevetoxin (Fig. 1(B) and datanot shown). The discovery of unsuspected interactions of reserpine/local anesthet-ics with brevetoxin sites on sodium channels reveals further facets of the com-plex modulatory aspects of toxin/drug interactions with this ion channel. Reser-pine appears to be a suitable probe for investigation of yet another modulatoryinteraction of agents with sodium channels and further insights should result fromelectrophysiological studies with combinations of batrachotoxin, brevetoxin, andreserpine.

ACKNOWLEDGMENT

The support of the NIH Undergraduate Scholarship Program for one of theauthors (K.O.) is gratefully acknowledged.

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reserpine: interactions with batrachotoxin and brevetoxin sites on voltage-dependent sodium channels

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