Proc. Nati. Acad. Sci. USA Vol. 83, pp. 189-192, January 1986 Neurobiology

Phencyclidine in low doses selectively blocks a presynaptic voltage-regulated potassium channel in rat brain (nerve terminals/synaptosomes/MRb efflux) D. K. BARTSCHAT AND M. P. BLAUSTEIN Department of Physiology, University of Maryland, School of Medicine, Baltimore, MD 21201 Communicated by Bernhard Witkop, September 5, 1985

ABSTRACT Phencylidine (PCP) is a major drug of abuse METHODS in the United States. It produces a toxic confusional psychosis in man. We show here that nanomolar to micromolar concen- Preparation of Synaptosomes. Synaptosomes prepared as trations of PCP and behaviorally active congeners selectively described (12, 14) were equilibrated with physiological salt block voltage-regulated noninactivating (or very slowly inac- solution (PSS; 5 mM KCI/145 mM NaCl/2 mM MgCl2/10 tivating) presynaptic K channels in the brain. The rank order mM glucose/0.5 mM Na2HPO4/0.1 mM unlabeled RbCl/10 of potency for blockage of these K channels parallels both the mM Hepes buffer titrated to pH 7.4 with NaOH) and were relative ability of these agents to produce characteristic behav- allowed to accumulate tracer IRb (20 ,Ci/ml of PSS; 1 Ci = ioral deficits in rats and their ability to displace [3H]PCP from 37 GBq) at 30'C for 30 min. its high-affinity binding sites in brain. In view of the enhanced Measurement of "Rb Efflux from Synaptosomes. Aliquots voltage-gated Ca influx that would be expected to accompany (30 1.l) of the 86Rb-loaded synaptosome suspension were blockage of presynaptic K channels, this mechanism could pipetted onto glass fiber filters, were washed free of extra- explain the excessive neurotransmitter release that is charac- cellular tracer with PSS, and were then exposed to efflux teristic of PCP intoxication. medium for various lengths of time (1-4 sec). Rb efflux was terminated by the rapid addition of "stopping solution" ion An understanding of the neuronal site(s) of action of containing the K-channel blockers tetraethylammonium phencyclidine [1-(l-phenylcyclohexyl)piperidine, PCP, "an- (145 mM), tetrabutylammonium ion (5 mM), and RbCl (0.1 gel dust"] is of major social importance, since PCP is widely mM) but no NaCl or KCl. Suction was rapidly applied, and abused in some areas of the United States. This drug the filters and filtrates were counted by liquid scintillation produces a toxic confusional psychosis in man that repro- spectroscopy; Rb efflux was expressed as: duces many ofthe primary symptoms of schizophrenia (1, 2). Rb efflux (%) = 86Rbffltrate x 100. The precise mechanism of action of this agent is not known, =6Rbfltrate + "Rbfilter but many studies suggest that the complex behavioral syn- The efflux medium was similar to the loading medium; in drome elicited by PCP is a consequence of altered central experiments involving increased external potassium concen- synaptic transmission (1, 2). While PCP binds with high tration, [K]0, K replaced Na mole-for-mole. When drugs affinity to brain membranes (3-5), the physiological activity were tested in the effiux medium, they also were added to the of these receptors has not been elucidated. wash medium to facilitate equilibration with the synapto- Several different mechanisms have been proposed to somes. See refs. 12 and 13 for additional details. account for the behavioral effects of PCP and its analogues: for example, they block the channels associated with nico- tinic cholinergic receptors (6, 7). However, many of the RESULTS analogues that display potent antinicotinic activity are be- Synaptosomes Possess Voltage-Regulated K Channels. Rb haviorally inactive (6, 7). Phencyclidines also block N- efflux from synaptosomes loaded with "Rb was used to methyl-D-aspartate (NMDA)-activated excitatory postsyn- assess the K permeability of the nerve terminals under aptic potentials in brain (8, 9), but this effect does not explain "resting" conditions and under conditions in which the the excess neurotransmitter release seen in PCP intoxication terminals were depolarized by increasing [K]0. 86Rb, with a (1, 2). In addition, PCP has been proposed to interact with a" half-life 36 times longer than 42K, is a suitable tracer for K (12) opiate receptors (4), but the existence of a distinct a opiate because (i) Rb, like K, is accumulated by synaptosomes via receptor subtype has been questioned recently (10). a metabolically active, ouabain-sensitive route; (it) Rb per- One effect of low concentrations of PCP is blockade of a meates most neuronal K channels nearly as well as does K portion of the K-stimulated 1Rb efflux from rat brain itself; and (iii) synaptosomes preloaded with both 42K and presynaptic nerve terminals (synaptosomes) (6, 11). We 86Rb have qualitatively similar K and Rb effluxes. recently examined the properties of "*Rb efflux from synap- K channel activity in synaptosomes was determined from tosomes and found four physiologically and pharmacologi- the Rb efflux as illustrated in Fig. 1. "Rb efflux under resting cally distinct K channels (12, 13). The experiments presented conditions (5 mM [K]o in Fig. 1) was about 0.3-0.4%/sec in this report were designed to determine whether PCP and (component R), which corresponds to a resting K permeabil- chemically related analogues selectively block a single class ity of 2.4 x 10-7 cm/sec (12). Rb efflux under these of nerve terminal K channels at concentrations relevant to conditions probably reflects the mechanism(s) responsible their behavioral effects. for the normal K permeability of the resting terminals.

The publication costs of this article were defrayed in part by page charge Abbreviations: PCP, phencyclidine [1-(1-phenylcyclohexyl)piperi- payment. This article must therefore be hereby marked "advertisement" dine]; [K]0, external potassium concentration; TCP, 1-[1-(2-thienyl)- in accordance with 18 U.S.C. §1734 solely to indicate this fact. cyclohexyl]piperidine. 189 Downloaded by guest on September 29, 2021 190 Neurobiology: Bartschat and Blaustein Proc. NatL Acad Sci. USA 83 (1986) I15 PCP Selectively Blocks Voltage-Regulated Noninactivating K Channels. To determine if PCP affects presynaptic K channels, this drug was tested for its ability to block the various components of the Rb efflux. In the experiment of I Fig. 1 Upper, the effects of 10 and 100 ,M PCP were examined in nominally Ca-free solutions. In 100 mM [K]0 medium, 10 AuM PCP depressed the 'Rb efflux through S by 04. about 35% but had negligible effect on component T. Increas- 0~~~~ .0 ing PCP to 100 ,uM had little additional effect on S but blocked component T by about 45%. Although not shown here, Tome,soecs component R and the Ca-dependent component C (13) were virtually unchanged by 100 ,M PCP. The dose-response curves for the effects of PCP on components S and Tare illustrated in Fig. 1 Lower. Note that low doses of PCP selectively blocked about one-third of 0 1 2 3 4 component S (Sv); higher doses inhibited T as well, but with Time,.sec little additional effect on S. Therefore, at PCP concentrations similar to those that elicit behavioral effects (0.1-1.0 mg/kg, 120 r corresponding to about 0.1-1 AM; ref. 15), the voltage- I Component T regulated presynaptic K channels, reflected in the PCP- sensitive portion of component S, should be selectively \o \ blocked. 80p- Is-o I The biphasic PCP dose-response curve for component Sin 0 Fig. 1 Lower implies that component S represents more than one class of K channels with different sensitivities to PCP. 601- 4-i This view is supported by calculations (12) that indicate that .0 depolarization of the terminals with 100 mM [K]0 should 401- Component S increase the driving force for unidirectional 'Rb efflux SR through the resting K permeability (component R), even with 20 no increase in conductance. Assuming ohmic behavior for R, about two-thirds ofthe increased Rb efflux observed between 0 1 and 4 sec in 100 mM [K]O (i.e., two-thirds of component S) can be accounted for by this electrodiffusion mechanism 0 -7 -5 (component SR in Fig. 1 Lower). The remaining Rb effiux PCP, log M through S (Sv) may be mediated by a class of voltage- regulated, noninactivating K channels that is uniquely sen- FIG. 1. Time course of 'Rb efflux from synaptosomes; the effect sitive to PCP. This view is supported by pharmacological of PCP. (Upper) 86Rb efflux in S mM K (o), 100 mM K (control) (o), experiments in which the inhibition of component S by 100 mM K/10 AtM PCP (*), or 100 mM K/100 AuM PCP (n). The data are means of six determinations. (Lower) PCP dose-response curves tetraalkylamines is evidently biphasic (12). for the inhibition of components S (o) and T(e). The data are means The possibility that component S consists of both Rb effiux offour determinations ± SEM. Similar results were obtained in three through PCP-sensitive voltage-regulated K channels (Sv) and other experiments. The components of 86Rb efflux indicated in the electrically driven efflux through voltage-independent com- figure are: R, Rb efflux in 5 mM K media (expressed in %/sec); S, ponent R (SR) was examined further in the experiments Rb efflux between 1 and 4 sec (%/sec) in K-rich medium minus represented in Fig. 2. The rate of "Rb efflux in nominally component R; T, K-dependent increment (%) in Rb efflux when the Ca-free media was measured at various [K]0 (see Fig. 1 efflux is extrapolated back to time zero; Sv = component of S that Upper) and figure 6 in ref. 12); the Rb efflux between 2 and is blocked by PCP; and SR = PCP-insensitive portion of component 4 sec (component 5) is plotted as a function of in Fig. 2 S. See the text and ref. 12 for additional [K]o details. (control). The Rb efflux increased sharply with increases in [K]o (i.e., with increasing depolarization) between 5 and 50 Depolarization of the synaptosomes with Ca-free 100 mM mM K and then leveled off. The expected contribution of R [K]O medium increased 'Rb efflux (Fig. 1, control); under to component S is shown as the dotted line in Fig. 2 (see ref. these conditions, two kinetically and pharmacologically dis- 12). When this experiment was repeated in the presence of tinct K permeabilities could be discerned. Between 1 and 4 100 &M PCP, the PCP-insensitive fraction of component S sec, the Rb efflux was linear and was 2.2-2.4%/sec (com- (SR in Fig. 2 legend) closely approximated the contribution ponent S). Extrapolation of the Rb efflux to the ordinate (zero expected from component R because of electrodiffusion time) exposed an additional, rapid component of the "Rb effects (Fig. 2, dotted line). This supports the view that two efflux (component 7). Component T reflects a distinct K K channels with different properties (SR and Sv) contribute channel that, unlike component 5, appears to inactivate in to component S and that Sv corresponds to a distinct class of less than 1 sec (12). voltage-regulated, noninactivating K channels that is sensi- Inclusion of Ca in the efflux medium enhanced Rb efflux in tive to PCP. 100 mM but not in 5 mM [K]O (data not shown). This Behaviorally Active Analogues of PCP Also Block Voltage- Ca-dependent "Rb efflux (component C) appears to be a Regulated, Noninactivating K Channels. If the behavioral manifestation of tetraethylammonium-sensitive, Ca-regulat- activity of PCP is related to its blockage of presynaptic K ed K channels (13). channels, PCP-like analogues should block these same K Pharmacological evidence indicates that components R, 5,' channels with a rank order of potency consistent with their T, and C correspond to distinct classes of K channels (12, 13). relative in vivo psychotomimetic activity. One of the most Component T is blocked by low concentrations of 4- potent behaviorally active PCP-like agents is TCP {1-[1-(2- aminopyridine; component C is selectively blocked by thienyl)cyclohexyl]piperidine; refs. 16 and 17}. TCP was micromolar quinine sulfate but not by 4-aminopyridine. found to be a more potent blocker of Sv than was PCP (Fig. Components R and S are much less sensitive to both drugs. 3 Upper). Fig. 3 Lower illustrates a test of the relative ability Downloaded by guest on September 29, 2021 Neurobiology: Bartschat and Blaustein Proc. Natl. Acad. Sci. USA 83 (1986) 191 Calculated depolarization, mV 100-0o O.. 0 OO .O. O. 0 10 20 30 40 50 60 70 U I I I I I I I 1 sv *4-O *g0 80- - 01"~~ S** - 3r r., I 0 c) '4-4 60 S 0 W) 0 I. 21 0~~~ el 1 ~~~~~~0 An_ :~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 0 0.* ..Io Q - 04 SR T co~ E 1 20 - 0 0 U ag0 O..""--@ oJ r I I I U I I U 0 0 -9 -8 -7 -6 -5 -4 -3 5 10 30 50 100 150 TCP, log M mM [K]0, 100 . FIG. 2. Effect of PCP on 'Rb efflux component S. The magni- tude ofcomponent S at the indicated [K]0 was expressed as the slope 80- 0Xv *0 of the least-squares line fitting the Rb efflux between 2 and 4 sec for 0.-%4X NO control (e) and 100 A&M PCP-containing solutions (o). The dotted line 0 Os,~~ is the expected contribution of the resting K permeability (R) to U- 60 - component S because of electrodiffusion effects (i.e., SR) when the 0 synaptosomes are depolarized to the levels indicated in the C) .A upper '4- abscissa (see the text and ref. 12). The values are the means of four 0 determinations. 40 c); of PCP and the chemically similar congeners m-amino-PCP 20 and m-nitro-PCP to block component S. The rank order of potency for these three drugs and TCP (Fig. 3 Upper) was: Hq? TCP > m-amino-PCP > PCP > m-nitro-PCP. A similar 0-I "I I I I sequence was observed when these agents were examined in 0 -7 -6 -5 -4 behavioral impairment paradigms (16, 17). In contrast, these Blocking agent, log M agents all blocked T only at higher concentrations, and they FIG. 3. Effect of PCP and related analogues on 86Rb efflux appeared to do so approximately equipotently [IC50s were components S and T. The experiments were carried out as described 100-500 /iM (Figs. 1 Lower and 3 Upper); other data not in the Fig. 1 legend. (Upper) Dose-response curve illustrating the shown]. effects of the PCP analogue TCP on components S (o) and T (o). The block of certain K channels by the PCPs may occur (Lower) Effects of PCP (A), m-amino-PCP (o), and m-nitro-PCP (0) from the intracellular side of the channel, since the mem- on component S. The data points are the means of two to six brane-impermeant methyl iodide derivative of PCP blocks individual experiments, each performed with four to six replicates. delayed rectification in muscle only when injected into the In two cases, m-amino-PCP (o), PCP (v), and m-nitro-PCP (e) were cells (18). The methyl iodide derivative of PCP had little all tested on the same synaptosome preparation, and the sequence of effect on component S in synaptosomes not relative potencies was as shown in Lower. The IC"0 values graphi- (data shown), cally determined by Eadie-Hofstee plots of individual experiments even though it displaced [3H]PCP from its high-affinity for the inhibition of component Sv were 0.02 and 0.05 AM for TCP, binding site in synaptic membranes (unpublished data). Thus, 0.3 and 0.1 gM for m-amino-PCP, 1.4 - 0.5 ,uM (SEM; n = 6) for it appears that blockage ofcomponent Sv by the PCPs results PCP, and 35 and 50 1.M for m-nitro-PCP. Other experiments, from interaction with the cytoplasmic face of presynaptic K performed under slightly different conditions, were consistent with channels. these values. Some workers have suggested that PCP interacts with cr DISCUSSION opiate receptors, since a agonists displace PCP from its Our data indicate that there may be a direct relationship high-affinity binding sites in brain (3, 4). We found (19) that between blockage of presynaptic voltage-regulated, nonin- certain oa opiates, which exist as stereoisomer pairs, also activating K channels and behavioral potency of the PCPs. selectively block the IRb efflux component Sv. However, This supports the view that these K channels may possibly be only the stereoisomer that produces PCP-like behavioral the primary sites of action of the PCPs in the brain. Our data effects was a potent blocker of Sv. This correlation between imply that the high-affinity PCP binding sites may be asso- the stereoselective behavioral potency and the K-channel- blocking activity provides further support for the hypothesis ciated with these channels and that PCP may be a useful that K channels may be involved in the psychotomimetic ligand to help identify and isolate these K channels. It should activity of both the PCPs and certain o- opiates. be noted that high-affinity PCP binding sites are nonhomo- The behavioral effects of PCP have been associated with geneously distributed in the brain (3-fold difference between excessive release of a wide variety of neurotransmitters: in regions with the highest and lowest densities ofbinding sites; particular, a massive dopamine release may underlie some of ref. 4), while the synaptosome preparation used in this study the most prominent symptoms of PCP intoxication (20). Our was prepared from whole forebrain. It will be of interest to results may explain the genesis of such an effect. By limiting determine whether the relative magnitude of Rb efflux the action-potential duration and regulating excitability, component Sv is enriched in various brain areas and whether presynaptic K channels are one of the primary factors that the distribution of these K channels parallels that of the influence Ca entry into nerve terminals and, consequently, high-affinity PCP binding sites. Ca-dependent transmitter release. Moreover, blockage of Downloaded by guest on September 29, 2021 192 Neurobiology: Bartschat and Blaustein Proc. Natl. Acad Sci. USA 83 (1986) only a fraction of presynaptic K channels may be sufficient 3. Vincent, J. P., Cavey, D., Kamenka, J. M., Geneste, P. & to prolong the action potential and produce derangement of Lazdunski, M. (1978) Brain Res. 152, 176-182. neurosecretion. It is plausible that such altered synaptic 4. Zukin, S. R. & Zukin, R. S. (1979) Proc. Natl. Acad. Sci. USA transmission at central synapses may underlie the 76, 5372-5376. disordered 5. Sorensen, R. G. & Blaustein, M. P. (1985) Biophys. J. 47, behavior characteristic of PCP intoxication. Therefore, our Supplement 2, 384a (abstr.). observations may provide a potential physiological link 6. Albuquerque, E. X., Aguayo, L. G., Warnick, J. E., between the binding of PCP to its high-affinity receptor in Weinstein, H., Glick, S. D., Maayani, S., Ickowicz, R. K. & brain and the ultimate behavioral effects ofPCP intoxication. Blaustein, M. P. (1981) Proc. Natl. Acad. Sci. USA 78, There is precedent for a modulatory role of K channels in 7792-7796. behavior. As an example, modulation ofa serotonin-sensitive 7. Albuquerque, E. X., Warnick, J. E., Aguayo, L. G., K current in Aplysia (the "S" current) is involved in simple Ickowicz, R. K., Blaustein, M. P., Maayani, S. & Weinstein, forms of learning in this marine mollusc (21). In mammalian H. (1983) in Phencyclidine and Related Arylhexylamines: Present and Future Applications, eds. Kamenka, J. M., Dom- systems, the K channel blocker 4-aminopyridine selectively ino, E. F. & Geneste, P. (NPP Books, Ann Arbor, MI), pp. blocks component T (12), prolongs nerve action potentials, 579-594. and enhances neurotransmitter release (22). Intoxication 8. Anis, N. A., Berry, S. C., Burton, N. R. & Lodge, D. (1983) with this agent in man may lead to dissociative behavior, Br. J. Pharmacol. 79, 565-575. agitation, confusion, convulsions, and coma (23). However, 9. Thomson, A. M., West, D. C. & Lodge, D. (1985) Nature the 4-aminopyridine-induced behavioral aberrations differ (London) 313, 479-481. qualitatively from those induced by PCP. This implies that 10. Quirion, R., O'Donohue, T. L., Everist, H., Pert, A. & Pert, blockage of various types of presynaptic K channels may C. B. (1983) in Phencyclidine and Related Arylhexylamines: modify integrated neuronal function and behavior; but the Present and Future Applications, eds. Kamenka, J. M., Dom- ino, E. F. & Geneste, P. (NPP Books, Ann Arbor, MI), pp. precise nature of the behavioral manifestations is likely to 667-684. depend upon the specific type of K channel that is affected. 11. Blaustein, M. P. & Ickowicz, R. K. (1983) Proc. Natl. Acad. In man, PCP intoxication causes a profound perceptual and Sci. USA 80, 3855-3859. cognitive disturbance that resembles the primary symptoms 12. Bartschat, D. K. & Blaustein, M. P. (1985) J. Physiol. 361, of schizophrenia (1, 2). An excess release of neurotransmit- 419-440. ters, especially dopamine, has been proposed to underlie the 13. Bartschat, D. K. & Blaustein, M. P. (1985) J. Physiol. 361, symptoms of both PCP intoxication (1, 2, 20) and schizo- 441-457. phrenia (24). While highly speculative, it is tempting to 14. Krueger, B. K., Ratzlaff, R. W., Strichartz, G. R. & consider the possibility (also see refs. 6, 7, and 11) that one Blaustein, M. P. (1979) J. Membr. Biol. 50, 287-310. as 15. Burns, R. S. & Lerner, S. E. (1981) in PCP (Phencyclidine): neuronal defect in some mental disorders such schizo- Historical and Current Perspectives, ed. Domino, E. F. (NPP phrenia may somehow involve a primary alteration in the ion Books, Ann Arbor, MI), pp. 449-469. channels responsible for the maintenance ofnormal electrical 16. Shannon, H. E. (1981) J. Pharmacol. Exp. Ther. 216, 543-551. activity in the nervous system. For example, a defect in K 17. Shannon, H. E. (1983) in Phencyclidine and Related channel activation could lead to prolongation of action Arylcyclohexylamines: Present and Future Applications, eds. potentials at nerve terminals and, thus, to excessive release Kamenka, J. M., Domino, E. F. & Geneste, P. (NPP Books, of a variety of neurotransmitters, including dopamine. Ann Arbor, MI), pp. 311-335. 18. D'Amico, G. A., Kline, R. P., Maayani, S., Weinstein, H. & B. A. B. K. Kupersmith, J. (1983) Eur. J. Pharmacol. 88, 283-290. We thank Drs. E. X. Albuquerque, Alger, Krueger, 19. Bartschat, D. K., Sorensen, R. G. & Blaustein, M. P. (1985) and R. G. Sorensen for helpful discussions and critical reading ofthe Soc. Neurosci. Abstr. 11, 316. manuscript. The work was supported by National Institutes of 20. Rappolt, R. T., Gay, G. R. & Farris, R. D. (1980) Clin. Health Grant NS 16106. Toxicol. 16, 509-529. 21. Klein, M. & Kandel, E. R. (1980) Proc. NatI. Acad. Sci. USA 1. Domino, E. F. & Luby, E. D. (1981) in PCP (Phencyclidine): 77, 6912-6916. Historical and Current Perspectives, ed. Domino, E. F. (NPP 22. Llinas, R., Walton, K. & Bohr, V. (1976) Biophys. J. 16, Books, Ann Arbor, MI), pp. 401-418. 83-86. 2. Petersen, R. & Stillman, R., eds. (1978) Phencyclidine (PCP) 23. Spyker, D. A., Lynch, C., Shobanowitz, J. & Sinn, J. A. Abuse: An Appraisal: Natl. Inst. on Drug Abuse Res. Mono- (1980) Clin. Toxicol. 16, 487-497. graph 21 (Government Printing Office, Washington, DC). 24. Snyder, S. H. (1981) Am. J. Psychiatry 138, 460-464. Downloaded by guest on September 29, 2021