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.