A Ca2+-channel blocker derived from funnelweb spider toxin (FIX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na'-dependent plateau potential. In the squid giant synapse, FIX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2l current and of transmitter release. FIX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10-to 12-pS conductance in 80 mM Ba2l and 5-8 pS in 100 mM Ca2+ with voltagedependent open probabilities and kinetics. High Ba2l concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2l. Both channels were blocked by FIX, Cd2+, and Co2l but were not blocked by w-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel.We propose that it be termed the "P" channel, as it was first described in Purkisje cells.While different Ca2l-channel blockers have been described in past years (1), a true blocker for the main Ca2+-dependent action potential in mammalian and in some molluscan neurons had not been encountered. This lack of a suitable blocker suggested that at least one of the main Ca2+ conductances present in the central nervous system (CNS) does not belong to the categories proposed by Tsien and co-workers (2, 3), as those channels respond quite specifically to dihyropyridines and w-conotoxins. The use of funnel-web spider toxin (FTX) as a CNS Ca2+-channel blocker was first described in Purkinje cells, where it blocked dendritic spiking (4). At that time the fragment utilized was known as AG1 and was obtained from Bioactives (Salt Lake City, UT). Because of the variability in the blocking observed, we attempted to isolate the main factor involved in this blockage from crude venom. We report here on the specific neuronal Ca2+-channel blocking properties of FTX and its use in the isolation of functional channels, which we have studied in the black lipid membranes. That the Ca2+-channel blocking fraction, which we call FTX, is different from that initially described as AG1 relates to its molecular mass as determined chromatographically, which is one-fifth that of AG1, and its high affinity for Ca2+ conduc...

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the a-conotoxin-and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channd (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N-or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K+-induced Ca'5 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FIX and sFVX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.Ca2' influx through voltage-dependent Ca2' channels (VDCCs) is the trigger for the release of neurotransmitters from the nerve terminals (1, 2). Three major types ofVDCC named T, L, and N were described in neuronal cells (3). The high-threshold L and N VDCCs are sensitive to the blocking effect of o-conotoxin (c-CgTX), and only the L type is affected by Ca2`channel antagonists of the 1,4-dihydropyridine (DHP) class. An intermediate-threshold VDCC channel called the P channel was identified in the; Purkinje cells of mammalian cerebellum and found to be insensitive to DHP and w-CgTX, but very sensitive to a low molecular weight fraction of the venom of the funnel-web spider Agelenopsis aperta (4). This funnel-web spider toxin (FTX) was also effective in blocking Ca2+ conductance and synaptic transmission at the squid giant synapse (4). Evoked release of neurotransmitter was shown to be dependent on Ca2+ influx through the N-type VDCC in sympathetic neurons by the inhibitory effect of w-CgTX and the lack of effect of DHP (5). By contrast, substance P release from dorsal root ganglia neurons (6, 7) and catecholamine release from chromaffin cells (8) are strongly inhibited by DHP, consistent with a major participation of L-type channels. However, mammalian motor nerve terminals are normally insensitive to either w-CgTX or DHP (9-11). Furthermore, in brain synaptosomes, K+-evoked Ca2' uptake and transmitter release are only partially sensitive to c-CgTX and DHP (12, 13). Thus the identity of the VDCC involved in transmitter release in the majority of the synapses at the mammalian central and peripheral nervous system has not been defined. The experiments presented here were designed to study the effect of FTX on transmitter release and Ca2+ influx at the mammalian neuromuscular junction and on Ca2+ uptake by cerebral cortex synaptosomes in order to determine whether a particular type of VDCC ...

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The distribution of the P-type calcium channel in the mammalian central nervous system has been demonstrated immunohistochemically by using a polydonal specific antibody. This antibody was generated after P-channel isolation via a fraction from funnel-web spider toxin (FIX) that blocks the voltage-gated P channels in cerebellar Purklije cells.In the cerebellar cortex, immunolabeling to the antibody appeared throughout the molecular layer, while all the other regions were negative. Intensely labeled patches of reactivity were seen on Purkije cell dendrites, especially at bifurcatlons; much weaker reactivity was present in the soma and stem segment. Electron microscopic loItion revealed labeled patches of plasma membrane on the soma, min_ dendrites, spiny branchlets, and spines; portions of the smooth endoplasmic reticulum were also labeled. Strong labeling was present in the periglomerular cells of the olfactory bulb and scattered neurons in the deep layer of the entorhinal and pyriform cortices. Neurons in the brainstem, habenula, nucleus of the trapezoid body and inferior olive and along the floor Of the fourth ventricle were also labeled intensely. Medium-intensity reactions were observed in layer H pyramidal cells of the frontal cortex, the CAl cells of the hippocampus, the lateral nucleus of the substantia nigra, lateral reticular nucleus,, and spinal fifth nucleus. Light labeling was seen in the neocortex, striatum, and in some brainstem neurons.Knowing the specific localization of voltage-gated ion channels on the soma-dendritic membrane of neurons is fundamental to understanding their intrinsic and integrative functions. The question of voltage-gated channel localiation has been of particular interest since the first report of dendritic action potentials in Purkinje cells over 2 decades ago (1, 2). Indeed, the existence of such electroresponsiveness was not readily accepted until intradendritic recordings were made from different points in the dendritic tree of Purkinje cells (3,4).The calcium-dependent nature of these potentials was initially shown in avians (5) and later in mammals (4,6). The recent availability of specific calcium-channel blockers has allowed a more precise identification of these conductances and the different types of calcium channels involved. Specifically, calcium-channel blockers such as the dihydropyridines (7, 8) and a-conotoxin (9, 10) were ineffective in Purkinje cells, while these responses were blocked by a funnel-web spider toxin (FTX) (11). The results obtained from the venom study were confirmed at both macroscopic current and single-channel levels for the calcium channels induced by rat brain mRNA injection into Xenopus oocytes (12, 13). The toxin (FTX) specifically responsible for this calcium-channel block was then isolated from the venom and a synthetic analog (sFTX) was made (14, 15). The calcium channel, called the P channel, was then isolated from bovine cerebella by using sFTX (16), and a polyclonal antibody was generated from this protein (17). In a more ...

Calcium plays several critical roles in the electrophysiology of mammalian central neurons. As a charge carrier, it is capable of generating either action potentials or graded voltage responses.' As a second messenger, calcium is involved in such events as transmitter release: the activation of ionic channels (e.g., the calcium-dependent potassium conductance'), and the phosphorylation of molecules, which in turn modulate ionic conductances.' Here we plan to review briefly some aspects of the voltagedependent calcium conductance in the neurons of the mammalian brain with particular emphasis on the conductance present in dendrites of cerebellar Purkinje cells.Before describing in detail the voltage-dependent channel properties of Purkinje cells, we will review in general terms the main calcium-dependent electroresponsiveness encountered in mammalian neurons. The first description of calcium-dependent spikes in vertebrate central nervous system (CNS) neurons was obtained by direct recordings from avian cerebellar Purkinje cell dendrites5 This was later confirmed in mammilary neurons.6 The existence of more than one voltage-dependent calcium conductance was originally encountered in the inferior olive (10). These cells demonstrated two types of responses, the high-and the low-threshold spikes (HTS and LTS, respectively).'.' Because the most common electroresponsiveness observed in intracellular recordings from different types of central neurons fall into these categories,' we will continue to describe them as stated above.By contrast the single channel responsible for the low-threshold calcium conductance was first described by Carbone and Lux? Later the calcium channels were grouped into three categories": (a) the T channels, which we now believe correspond to the low-threshold calcium conductance; (b) the N channels, which correspond to a certain extent to the high-threshold calcium conductance; and (c) the L calcium channel, which does not seem to be very commonly represented in the CNS, but which would also fall into this category of high-threshold calcium conductance. A simple incorporation of our nomenclature into the framework proposed above"' may prove to be problematic because the criteria for these two characterizations are so different. The single-channel criteria are based on direct measurements and do not consider cable properties. Ours was developed from the standpoint of neuronal integration, which takes into account parameters such as the spatial location of the 103

The effect of the IgG from amyotrophic lateral sclerosis (ALS) patients was tested on the voltagedependent barium currents (IB.) Amyotrophic lateral sclerosis (ALS; "Lou Gehrig's disease") has been proposed to be an autoimmune disease that can attack upper and lower motoneurons (1), leading to paralysis, respiratory depression, and death. It has been previously reported that IgG from ALS patients has a blocking effect on L-type calcium channels (2) and in lipid bilayer (3). In addition, ALS IgG enhances calcium currents (Ica) in immortalized motoneurons (4), acting on an as yet unidentified calcium channel. This IgG has also been shown to produce an increase in acetylcholine release (5), a mechanism in which the P-type calcium channel is directly involved (6). Here, we demonstrate, using patch-clamp recordings from Purkinje cells (7,8), that ALS IgG produces an increase in barium current (IB.). A parallel experiment using purified P-type calcium channels isolated from the cerebellum and incorporated into a lipid bilayer (9,10)

1. The effects of the calcium channel blockers, funnel‐web spider toxin (FTX), omega‐agatoxin IVA (omega‐Aga IVA) and omega‐conotoxin GVIA (omega‐CgTX), were tested on transmitter release and presynaptic currents in frog motor nerve endings. 2. Evoked transmitter release was blocked by FTX (IC50 = 0.02 microliter ml‐1) and omega‐CgTX (1 microM) but was not affected by omega‐Aga IVA (0.5 microM). When FTX (0.1 microliter ml‐1) was assayed on spontaneous release either in normal Ringer solution or in low Ca(2+)‐high Mg2+ solution, it was found not to affect miniature endplate potential (MEPP) amplitude but to increase MEPP frequency by approximately 2‐fold in both conditions. 3. Presynaptic calcium currents (ICa), measured by the perineurial technique in the presence of 10 mM tetraethylammonium chloride (TEA) and 200 microM BaCl2 to block K+ currents, were blocked by omega‐CgTX (5 microM), partially blocked by FTX (1 microliter ml‐1) and not affected by omega‐Aga IVA (0.5 microM). 4. The presynaptic calcium‐activated potassium current (IK(Ca)) measured by the perineurial technique in the presence of 0.5 microM 3,4‐aminopyridine (DAP) to block voltage‐dependent K+ currents, was strongly affected by charybdotoxin (ChTX) (300 nM) and completely abolished by BaCl2 (200 microM). This current was also blocked by omega‐CgTX (5 microM) and by CdCl2 (200 microM) but was not affected by FTX (1 microliter ml‐1). The blockade by omega‐CgTX could not be reversed by elevating [Ca]o to 10 mM. 5. The results suggest that in frog synaptic terminals two omega‐CgTX‐sensitive populations might coexist. The transmitter release process seems to be mediated by calcium influx through a omega‐CgTX‐ and FTX‐sensitive population.