Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 4538-4542, June 1990 Neurobiology Funnel-web spider venom and a toxin fraction block calcium current expressed from rat brain mRNA in Xenopus oocytes J.-W. LIN, B. RUDY, AND R. LLINAS Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue, New York, NY 10016 Contributed by R. Llinds, March 12, 1990

ABSTRACT Iijection of rat brain mRNA into Xenopus studies have revealed Ca channels that do not conform to the oocytes has been shown to induce a calcium current ('Ca) that existing three types. Studies of calcium flux in brain synap- is insensitive to dihydropyridine and c-conotoxin. We exam- tosomes revealed a combination of kinetic and pharmaco- ined the effect offunnel-web spider venom on two aspects ofthis logical properties that did not fit L-, N-, or T-type behavior expressed ICa: (i) the calcium-activated chloride current [Ic'(ca)] (11, 12). Dendritic calcium spikes of cerebellar Purkinje cells and (it) the currents carried by barium ions through calcium (13) and Ca currents expressed in Xenopus oocytes from rat channels (IB,). In the presence of 1.8 mM extracellular calcium, brain mRNA are totally insensitive to DHP or w-conotoxin 'CI(Ca) tail current became detectable between -30 and -40 mV (14), whereas both currents have N-type kinetics-i.e., un- from a holding potential of -80 mV and reached a maximal detectable until -40 mV and showed little inactivation. Thus, amplitude between 0 and +10 mV. Total spider venom partially a broader classification scheme is needed to incorporate (83%) and reversibly blocked the calcium-activated chloride these channel types, and the identification of new pharma- current without changing its voltage sensitivity. A chromato- cological tools to differentiate Ca channels has become an graphic toxin fraction from the venom also blocked this current urgent task. (64%). The venom had a minimal effect on IN. and IK. Direct A fraction of funnel-web spider venom has been charac- investigation of inward current mediated by calcium channels terized recently (15) and shown to block the calcium current was carried out in high-barium solution. IBa had a higher threshold of activation (-30 to -20 mV) and reached its in the squid giant synapse and calcium spikes in cerebellar maximal amplitude at about +20 mV. Total venom or a partly Purkinje cells (13, 16). Because the calcium channels in both purified chromatographic toxic fraction blocked 1B. partially preparations are insensitive to DHP and w-conotoxin and and reversibly without changing its current-voltage character- have a threshold higher than T-type channels, these venom- istics. Furthermore, the extent of the total venom block de- sensitive channels have been assumed to represent another pended on the concentration ofextracellular barium. Only 35% class and have been named P (for Purkinje cells) channels of the 1B. was blocked in 60 mM Ba2+, whereas the block (13). Calcium channels of similar characteristics have also increased to 65% and 71%, respectively, for 40 and 20 mM been observed in neurohypophysis and cerebellar granular Ba2+. On the basis of these results, we propose that the calcium cells (17, 18). channels expressed from rat brain mRNA in Xenopus oocytes is In this report, we characterize the effect of the funnel-web similar to the recently discovered P-type channels. spider venom and its toxin fraction (FTX) on the Ca current expressed from rat brain mRNA in Xenopus oocytes. Our The role of calcium channels in central nervous system results show that they can block the expressed Ca current, neurons is important and diverse. Somadendritic calcium and the level of the block depends upon the concentration of currents control firing patterns by way of rebound activation divalent cations extracellularly. of low-threshold currents (1, 2) or by the activation of calcium-dependent potassium currents (3). Dendritic calcium METHODS channels provide local active responses and expand the complexity of neuronal integration (4). Calcium currents Adult Xenopus laevis were maintained in fresh water (200C) located in the presynaptic terminals are essential for trigger- and fed weekly. Surgical removal of oocytes was performed ing transmitter release (5, 6). Finally, calcium influx through under anesthesia (0.17% MS222). After isolation, oocytes voltage-sensitive calcium channels can play the role of a were treated with collagenase (2 mg/ml, Sigma type 1A) second messenger, by triggering protein phosphorylation (7) dissolved in OR(2) (82.5 mM NaCl/2.0 mM KCl/1.0 mM or the inositol trisphosphate pathway (8), and produce long- MgCl2/5.0 mM Hepes, titrated to pH 7.4). Collagenase was lasting effects on neuronal behavior. These functions are washed out after 45-60 min. The oocytes were then selected probably mediated by various types of Ca channels and [stage V and VI (19)] and transferred to PS solution (96 mM require their strategic distribution on the plasma membrane. NaCl/2.0 mM KCl/1.8 mM CaCl2/1.0 mM MgCl2/5.0 mM Present classification of mammalian central nervous sys- Hepes/2.5 mM pyruvate, pH 7.4, containing penicillin at 100 tem neuronal calcium channels, which includes L, N, and T units/ml and streptomycin at 100 gg/ml). Injection of RNA types, is based on physiological and pharmacological criteria [50 nl of RNA (1 ,ug/,ul) per oocyte] was carried out 24 hr established from studies of dorsal root ganglion neurons (2). later, and oocytes were maintained in PS solution thereafter. Following this classification, the L-type, dihydropyridine Electrophysiological study of the oocytes was performed (DHP)-sensitive channels have been demonstrated in disso- 48-96 hr after the injection. ciated hippocampal neurons (9), whereas low-threshold cal- RNA Preparation. Whole brain RNA was isolated from cium current recorded from inferior olivary and thalamic 16-day-old rats after the procedure of Dierks et al. (20). neurons corresponds to T-type channels (10). However, the Poly(A)+ RNA was purified from total RNA on oligo(dT)- classification appears to be too restricted because many Abbreviations: FTX, funnel-web spider toxin fraction; DHP, dihy- The publication costs of this article were defrayed in part by page charge dropyridine; I, current; V, voltage; ICa, 'Ba' INa, IK, currents of payment. This article must therefore be hereby marked "advertisement" calcium, barium, sodium, and potassium, respectively; ICI(ca)' cal- in accordance with 18 U.S.C. §1734 solely to indicate this fact. cium-activated chloride current. 4538 Downloaded by guest on September 28, 2021 Neurobiology: Lin et al. Proc. Natl. Acad. Sci. USA 87 (1990) 4539 cellulose type III (Collaborative Research) according to activated by calcium channels intrinsic to the oocytes and/or established protocols (21). the calcium channels synthesized from the injected RNA. Electrophysiology. Two-electrode voltage clamp was used The contribution of the native calcium current to ICI(Ca) is to characterize the currents mediated by ion channels ex- small because the amplitude of this current in noninjected pressed from injected mRNA. Both voltage recording and oocytes was 10 to 100 times smaller than that recorded in current electrodes were filled with 3 M KCl when lcl(ca) was injected oocytes. The oocytes were typically held at -80 mV studied. In those experiments where barium currents were and depolarized in 10-mV increments by 400-msec pulses. isolated, current electrodes were filled with a 1:1 mixture of Depolarizations above -30 to -40 mV started to activate a 3 M tetraethylammonium chloride/3 M cesium chloride. The tail current after termination of the voltage steps (Fig. LA). current electrode holder was attached to a pressure source to Because the holding potential was near EK, the tail current inject K channel blockers. Electrode resistance ranged from mostly reflected the ICI(Ca) activated by the depolarizing 0.5-2 Mfl. pulses. Furthermore, previous studies have demonstrated Typically, the oocytes were held at -80 mV and stepped that ICI(Ca) was not voltage dependent in the potential range up to +50 mV in 10-mV increments; the pulse duration was investigated here (24). Thus, a plot of the tail-current ampli- 400 msec. For analysis of the calcium-activated chloride tude against the voltage steps activating it provides an current, the recordings were obtained in ND96 (ND96 is indication on the voltage sensitivity of the underlying Ca identical to PS solution except that penicillin, streptomycin, channels. In the I-V plot shown in Fig. 1B, this Cl tail current and pyruvate were omitted). To study calcium current in became visible between -30 to -40 mV and reached peak isolation, the extracellular solution was replaced by high-Ba amplitude at 0 mV. Further depolarization led to a reduction and Cl-free solution (BaMS: 60 mM Ba(OH)2/20 mM NaOH/ of this current. The tail current and the outward current 2 mM KOH/5 mM Hepes, titrated to pH 7.4 with methane- during the pulse (Fig. LA) were partially and reversibly sulfonic acid). Furthermore, 1 AM tetrodotoxin was used to blocked when the crude venom was washed into the record- block INa, and 10 mM tetraethylammonium chloride was ing chamber. Most, if not all, of the remaining outward added to the bath to block IK and to stabilize the bath current during the pulse was probably due to expressed K potential. Pressure injection of the tetraethylammonium/ channels that were insensitive to the venom. The block was cesium mixture from the current electrodes also facilitated IK a simple reduction of the current amplitudes, whereas the blockade. In some experiments, the Ba2+ concentration was shape of the I-V curve remained unchanged (Fig. 1B). On varied systematically; the detailed ionic compositions of average 83% (n = 13) of the 'CI(ca) was blocked by a 1:1000 these solutions are specified in the figure legends. Data dilution of the crude venom, and higher concentrations did acquisition and analysis were performed with the pClamp not produce additional block. Similar results were obtained system (Axon Instruments, Burlingame, CA). When IBa was when FTX was used (64% block, n = 5). The effect of the studied, four to eight traces were routinely averaged to spider venom on INa and IK seemed minimal because the improve the signal-to-noise ratio. All experiments were per- amplitude and waveform of both currents were changed little formed at a bath temperature of 18-20°C. after the toxin (25). The reduction of ICI(Ca) is not due to the Crude spider venom (Agelenopsis aperta or Hololena effect of the venom on the Cl channels directly because curta) or toxin partially purified by chromatography (FTX, pressure injections of Ca could evoke this current in com- from A. aperta) (13) was applied to the oocytes by perfusion. parable amplitudes before and after the toxin (25). Therefore, The volume of the chamber was 300 to 400 ,l, and the effect the venom blocked ICi(ca) reversibly by interfering with the of the toxin was recorded after 5-8 ml of the solution underlying Ca channels. containing venom was washed in. Unless otherwise indi- The spider venom blocks ICI(Ca) in a dose-dependent man- cated, experiments illustrated in the Results section were ner. The I-V plot in Fig. 2A shows that ICI(Ca) was blocked to obtained with the crude venom. different levels in the presence of two toxin concentrations. In this case, dose 1 was equivalent to a 1 ,ul/ml dilution of FTX purified chromatographically, and higher concentra- RESULTS tions generally did not produce further block. A dose- Effect ofthe Spider Venom on ICI(ca). The effect ofthe spider response relationship obtained from a similar experiment is venom on the 'CICa, was studied in ND96 solution. The Ici(Ca) shown in Fig. 2B. The fitting curve was drawn by hand and is an intrinsic current ofXenopus oocytes (22, 23) and can be does not imply any binding characteristics. With a 10-fold A B V (mV) -100 -50

a control & FTX * wash < HoIC 100 msec (nA) L 500

FIG. 1. Spider venom blocks ICI(ca). (A) Voltage-activated currents recorded before and after the venom. These currents were activated by voltage pulses stepped to 0 mV from a holding potential of -80 mV. After application of total spider venom (1:1000 dilution), the chloride component of the outward current is blocked, leaving the IK component. The blocking effect is clearer for the tail current, where there is little contamination of potassium current. Washing out the venom for 1 hr resulted in partial recovery. (B) I-V plot Of ICl(Ca). Tail current amplitudes were plotted against voltage steps. Maximal Icl(ca) tail was activated by pulses stepped to 0 mV. The venom reduced the current amplitude without changing the voltage sensitivity. The plot was obtained from the same oocytes used in A. Downloaded by guest on September 28, 2021 4540 Neurobiology: Lin et al. Proc. Natl. Acad. Sci. USA 87 (1990) A B mV 1.0- -50 50 0.8- 0~~~ .0 0 0.6- - 0 a :Control c -I. 0.4 0~~~ * :2/5 C 0 x :1 0 0.2-

I 0.04 0.0 .01 .1 1 Dose

FIG. 2. Dose-dependent block ofICI(ca) by FTX. (A) I-V plot OfICI(Ca) under control conditions and after application of two concentrations ofpartially purified toxin. The maximal quantity oftoxin added to the 2-ml bath was 2 jl; the other dose was 2/5ths ofthis maximal concentration. Note that the I-V characteristics were independent of the level of the block. All data were collected from the same oocyte. We started with the lowest concentration and increased the concentration by adding venom without washing. Recordings were collected at least 15 min after each dose was added. (B) Dose-dependent block ofmaximal ICI(ca) measured at 0 mV. The x axis corresponds to concentrations of FTX in ;J/ml. Percentage of remaining current was normalized by the current measured before addition of any toxin. increase in the FTX concentration (right half of the graph), curve of Ici(ca) shown in Fig. 1B is due to high concentration the remaining current decreased from 85 to 10%. of divalent cations (26) because a similar shift was seen for Effect of the Spider Venom on IBa* The direct effect of the ICI(Ca) when extracellular calcium concentration was raised spider venom on ICa was examined by isolating inward from 1.8 to 15.6 mM (data not shown, see also ref. 23). The currents in a high-barium solution where Cl- was replaced by application of FTX, at 1:1000 dilution, reversibly reduced the methanesulfonate and tetrodotoxin and tetraethylammonium amplitude ofIBa (Fig. 3A), whereas the waveform and voltage were added to block Na and K channels. Under these sensitivity of the remaining currents were not affected (Fig. conditions, the only inward current will be carried by Ba ions 3). Because the 'Ba block was noted to be less extensive than moving through Ca channels. Depolarizing pulses activated that seen for ICI(Ca) (Fig. 1B, for example), we examined the an inward current showing little inactivation (Fig. 3A). This possible effect of Ba2+ concentration on this block. Signifi- current started to appear at -20 mV and reached peak cant changes in 'Ba amplitude were seen when the extracel- amplitude at about +20 mV (Fig. 3 A and C). The rightward lular Ba2+ concentration was varied (Fig. 4A). The peak shift of the voltage sensitivity in comparison with the I-V inward current in 20 mM Ba2+ was -40% of that recorded in A control FTX wash

100 mec I °C

B C V (mV) -100 -50 0 50 10 mV

I-N &I FTX k wash = control -400 n FTX control * wash

200n A (nA) C 500msec -800 \0

FIG. 3. Barium current is partially and reversibly blocked by FTX. (A) Examples of an IBa series recorded before and after FTX and after washout. Inward current started to appear between -20 to -30 mV and generally showed little inactivation. (The slight decline of the larger currents during the pulses was probably from incomplete block ofIK.) Applications ofthe toxin reduced the current amplitudes without changing current waveform. One-hour wash partially recovered the toxin-blocked currents. (B) Superimposed traces to provide direct comparison of control, blocked, and recovered currents. These traces were activated by voltage pulses stepped to +10 mV. (The residual current and leakage were corrected by subtracting residual currents measured in 100 ,um Cd.) (C) I-V plot of IBa obtained from the same oocyte as in A and B. The toxin reduced the current without changing the voltage sensitivity of the Ca channels. Downloaded by guest on September 28, 2021 Neurobiology: Lin et al. Proc. Natl. Acad. Sci. USA 87 (1990) 4541 A B 20 mM -10mV 100

+10mV 00°3- N co 80 40 mM 0 z0 . 60

60 mM cts 40 m / 5 20 40 60 100 msec [Ba]o (mM) FIG. 4. IBa dependence on extracellular Ba2+ concentration ([Bal]). (A) IBa recorded from the same oocyte in three different Ba2+ concentrations. Each pair ofcurrents was activated by voltage step to -10 and +10 mV. LargerIBa was recorded as [Ba]0 was increased. There is a more apparent decline in IBa in low [Ba]0, (compare the + 10 mV traces of20 mM and 60 mM Ba2+ results); this is probably from less complete block of IK in 20 mM Ba2+ rather than from inactivation of Ca channels. (B) [Ba]0 dependence of barium current amplitude. Data points were pooled from four oocytes, and each oocyte was exposed to several Ba2+ concentrations. Currents were normalized by the maximal current recorded in 60 mM Ba2+ for individual oocytes before the data were pooled for this graph. The maximal current occurred at different voltages as [Ba]0 was changed; normally there is a shift of 20-30 mV to the right as [Ba]0 was increased from 10 to 60 mM. The ionic composition of 20 and 40 mM Ba2+ solutions are as follows: 20 mM Ba solution-20 mM Ba(OH)2/50 mM NaOH/2 mM KOH/5 mM Hepes/30 mM tetraethylammonium hydroxide, titrated to pH 7.4 with methanesulfonic acid; 40 mM solution-40 mM Ba(OH)2/20 mM NaOH/2 mM KOH/5 mM Hepes/30 mM tetraethylammonium hydroxide, titrated to pH 7.4 with methanesulfonic acid. 60 mM Ba2+ (Fig. 4B). A rightward shift of the voltage steps 20 mM Ba2+, and 65% and 35% of the current were blocked that activated maximal current was seen as Ba2+ concentra- in 40 and 60 mM Ba2+, respectively. tion was increased (data not shown). The same concentration of the crude venom was less effective in blocking IBa in 60 mM Ba2+ than in lower Ba2+ DISCUSSION concentrations. The examples illustrated in Fig. 5 A-C show The characterization of mammalian central nervous system that the total venom blocked most 'Ba in 20 mM Ba2+ (A), Ca channels is clearly just beginning. The study of induced whereas only 30% of the current was blocked in 60 mM Ba2+ channels in Xenopus oocyte circumvents major technical (C). Results obtained from 22 oocytes are pooled in Fig. SD difficulties associated with the accessibility ofion channels in where, on average, the spider venom blocked 71% of IBa in dendrites and synaptic terminals. However, the application A B 20 mM 40 mM

I1< c To

c D 80 60 mM in-5

lTn-5 60 0 4o I0- 40 \4n-12

50 msec 20 40 60 [Ba]o (mM) FIG. 5. [Ba]0 dependence ofthe venom blocks. (A-C) Barium current blocked by the venom under different [Ba]0 levels. Each pair of traces shows 'Ba before and after blockade. Recordings were obtained from three different oocytes, and the traces shown here were the maximal current of each voltage clamp series: 0 mV for 20 mM, and 20 mV for 40 mM and for 60 mM Ba2+. Most of the inward current was blocked in 20 mM Ba2+ (A), whereas only a 30%o reduction occurred in 60 mM Ba2+ (C). (D) Effect of [Ba]0 on venom potency at 1 ,ul/ml concentration. About 71% Of'Ba was blocked by the venom in 20 mM Ba2+, whereas only 65% and 35% ofthe current was blocked in 40 and 60 mM Ba2+, respectively. Sample size and SD are indicated. Data were collected from 22 different oocytes, and percentages of blocks were calculated from the voltage steps that activated maximal current. Downloaded by guest on September 28, 2021 4542 Neurobiology: Lin et al. Proc. Natl. Acad. Sci. USA 87 (1990) of this approach to Ca channels is still in an early stage. binding curve for calcium channels than Ba ions have for the Studies of Ca channels expressed from human temporal ion permeation pore. cortex mRNA demonstrated a single ICa component that is w-conotoxin sensitive (27). In contrast, Leonard et al. (14) This work was supported by Grant NS13742 from the National reported that rat brain mRNA-induced 'Ca was insensitive to Institute ofNeurological and Communicative Disorders and Strokes, DHP or w-conotoxin (however, see ref. 28). Our results by the Fidia Research Foundation for funding and venom, and by demonstrate that a toxin present in Agelenopsis spider venom Grant 26976 from the National Institute ofGeneral Medical Sciences. blocks the calcium currents expressed in Xenopus oocytes 1. Llinas, R. & Yarom, Y. (1981) J. Physiol. (London) 315, after injection ofrat brain mRNA. Because the native venom 569-584. and the toxin fraction FTX have similar blocking properties, 2. Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature we will assume for the purpose of this discussion that FTX is (London) 316, 440-443. the calcium channel-blocking agent. 3. Lancaster, B. & Nicoll, R. A. (1987) J. Physiol. (London) 389, On the basis of its abundance of expression and its similar 187-203. slow inactivation kinetics to that measured by Ca flux studies 4. Llinds, R. & Sugimori, M. (1980) J. Physiol. (London) 305, ofbrain synaptosomes (29), Leonard et al. (14) suggested that 171-195. the calcium channels expressed in the oocytes injected with 5. Katz, B. (1969) The Release ofNeural Transmitter Substances rat brain mRNA could mediate neuronal transmitter release. (Thomas, Springfield, IL). 6. Llin.s, R., Steinberg, I. Z. & Walton, K. (1981) Biophys. J. 33, However, review of published studies on the spider toxin 323-352. indicated that the toxin-sensitive calcium channels play func- 7. Nestler, E. J. & Greengard, P. (1984) Protein Phosphorylation tional roles broader than simply triggering transmitter re- in the Nervous System (Wiley, New York). lease. Calcium spikes in the dendrites of cerebellar Purkinje 8. Eberhard, D. A. & Holz, R. W. (1988) Trends NeuroSci. 11, cells exhibit identical pharmacological and electrophysiolog- 517-520. ical properties-i.e., insensitive to DHP or c-conotoxin, 9. Doerner, D., Pitler, T. A. & Alger, B. E. (1988) J. Neurosci. 8, block by FTX, and slow inactivation kinetics (13). Additional 4069-4078. examples of the spider venom-sensitive calcium current 10. Llinas, R. (1988) Science 242, 1654-1664. include the following: (i) the presynaptic calcium current in 11. Woodward, J. J., Rezazadeh, S. M. & Leslie, S. W. (1988) the squid giant synapse (13); (it) presynaptic calcium spikes, Brain Res. 475, 141-145. measured the 12. Suszkiw, J. B., Murawsky, M. M. & Shi, M. (1989) J. Neuro- optically, of frog neurohypophysis (17); (iii) chem. 52, 1260-1269. soma-dendritic calcium current in cerebellar granular cells in 13. Llinas, R., Sugimori, M., Lin, J.-W. & Cherksey, B. (1989) tissue culture (18); and (iv) calcium currents measured in Proc. Natl. Acad. Sci. USA 86, 1689-1693. Drosophila neurons (30). Among the examples listed, when 14. Leonard, J. P., Nargeot, J., Snutch, T. P., Davidson, N. & ICa was measured, the spider venom-sensitive calcium cur- Lester, H. A. (1987) J. Neurosci. 7, 875-881. rents all exhibited a similar noninactivating kinetics (howev- 15. Cherksey, B., Llinds, R., Sugimori, M. & Lin, J.-W. (1990) er, see ref. 31). Therefore, although the spider venom- Biol. Bull. 177, 321. sensitive channels studied thus far may all belong to the 16. Sugimori, M. & Llinas, R. (1987) Soc. Neurosci. Abstr. 13, 228. P-channel family, their distribution and functional roles may 17. Salzberg, B. M., Obaid, A. L., Stanley, K., Lin, J.-W., Sugi- mori, M., Cherksey, B. & Llinis, R. (1990) Biophys. J. Abstr. be diverse. 57, 305a. We found that the spider toxin block ofthe calcium current 18. Bertolino, M., Vicini, S., Costa, E. & Llinas, R. (1990) FASEB expressed from rat brain RNA depends upon the concentra- J. Abstr. 4, A1202. tion of Ba ions. The toxin may block Ca channels by plugging 19. Dumont, J. N. (1972) J. Morphol. 136, 153-180. the permeation pore. In this case, the toxin and Ba2+ would 20. Dierks, P., van Ooyen, A., Mantei, N. & Weissmann, C. (1981) compete for a site at the channel mouth. Alternatively, Ba2+ Proc. Natl. Acad. Sci. USA 78, 1411-1415. may screen negative charges at or close to the venom-binding 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular sites and compete with its binding, such as in the competition Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold between charybdotoxin and monovalent cations for calcium- Spring Harbor, NY). These sites not be 22. Miledi, R. (1982) Proc. R. Soc. London Ser. B 215, 491-497. activated potassium channels (30). need 23. Barish, M. E. (1983) J. Physiol. (London) 342, 309-325. the permeation pore itself, and the toxin may block either by 24. Miledi, R. & Parker, I. (1984) J. Physiol. (London) 357, a plugging mechanism or by modulating the gating of the Ca 173-183. channels. The divalent cation dependence may also explain 25. Lin, J.-W., Rudy, B., Cherksey, B., Sugimori, M. & Llinas, R. why the native venom or FTX block was never complete, (1989) Soc. Neurosci. Abstr. 15, 652. either in ND96 or in high Ba2+. This may simply be due to a 26. Hille, B., Woodhull, A. M. & Shapiro, B. I. (1975) Philos. baseline competition between the toxin and existing divalent Trans. R. Soc. London Ser. B 270, 301-318. cations in the solution. A related observation is that changing 27. Gundersen, C. A., Umbach, J. A. & Swartz, B. E. (1988) J. the divalent cation concentration had a differ- Pharmacol. Exp. Ther. 247, 824-829. quantitatively 28. Kaneko, S. & Nomura, Y. (1987) Neurosci. Lett. 83, 123-127. ent effect on the spider venom block and the IBa. Specifically, 29. Suszkiw, J. B., O'Leary, M. E., Murawsky, M. M. & Wang, the level ofblock was reduced to halfwhen Ba2+ was changed T. (1986) J. Neurosci. 6, 1349-1357. from 40 to 60 mM (Fig. 5). In contrast, there was only a 20% 30. Anderson, C. S., MacKinnon, R., Smith, C. & Miller, C. (1988) change in the 'Ba amplitude for the same concentration range J. Gen. Physiol. 91, 317-333. (Fig. 4). This behavior suggests that, regardless of where the 31. Leung, J.-T., Branton, W. D., Phillips, H. S., Jan, L. & binding site is located, the spider toxin has a shallower Byerly, L. (1989) Neuron 3, 767-772. 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