The C terminus of the Ca channel alpha1B subunit mediates selective inhibition by G-protein-coupled receptors.

Inhibition of calcium channels by G-protein-coupled receptors depends on the nature of the Galpha subunit, although the Gbetagamma complex is thought to be responsible for channel inhibition. Ca currents in hypothalamic neurons and N-type calcium channels expressed in HEK-293 cells showed robust inhibition by G(i)/G(o)-coupled galanin receptors (GalR1), but not by Gq-coupled galanin receptors (GalR2). However, deletions in the C terminus of alpha(1B-1) produced Ca channels that were inhibited after activation of both GalR1 and GalR2. Inhibition of protein kinase C (PKC) also revealed Ca current modulation by GalR2. Imaging studies using green fluorescent protein fusions of the C terminus of alpha(1B) demonstrated that activation of the GalR2 receptor caused translocation of the C terminus of alpha(1B-1) to the membrane and co-localization with Galphaq and PKC. Similar translocation was not seen with a C-terminal truncated splice variant, alpha(1B-2). Immunoprecipitation experiments demonstrated that Galphaq interacts directly with the C terminus of the alpha(1B) subunit. These results are consistent with a model in which local activation of PKC by channel-associated Galphaq blocks modulation of the channel by Gbetagamma released by Gq-coupled receptors.

Characteristics of voltage sensitive calcium channels in dendrites of cultured rat cerebellar neurons.

We examined the effects of various pharmacological agents on the Ca2+ channels existing in the dendrites of cultured rat cerebellar neurons. The cultures consisted of Purkinje cells and non-Purkinje cells (deep cerebellar nuclear neurons and other non-granule neurons). Changes in intracellular free calcium concentration, [Ca2+]i, were monitored by digital imaging microfluorimetry using fura-2 as the indicator dye. In the Purkinje cell population increases in dendritic [Ca2+]i evoked by brief pulses of high K+ were very effectively blocked by (> 80%) by omega-Aga-IVA at low concentrations. Nimodipine and omega-conotoxins GVIA and MVIIC only blocked small components of the [Ca2+]i rise. In the non-Purkinje cells, omega-Aga-IVA was much less effective. omega-Conotoxin-GVIA blocked somewhat more and nimodipine blocked a similar percentage of the [Ca2+]i rise. omega-Conotoxin-MVIIC was quite ineffective in these cells. The results are discussed in terms of the types of voltage sensitive Ca2+ channels existing in the dendrites of these cells.

Maturation of vulnerability to excitotoxicity: intracellular mechanisms in cultured postnatal hippocampal neurons.

Neuronal vulnerability to excitotoxicity changes dramatically during postnatal maturation. To study the intracellular mechanisms by which maturation alters vulnerability in single neurons, we developed techniques to maintain hippocampal neurons from postnatal rats in vitro. After establishing their neuronal phenotype with immunohistochemistry and electrophysiology, we determined that these neurons exhibit developmentally regulated vulnerability to excitotoxicity. At 5 days in vitro, NMDA-induced cell death at 24 h increased from 3.6% in 3-day-old rats to >90% in rats older than 21 days. Time-lapse imaging of neuronal morphology following NMDA demonstrated increasingly prevalent and severe injury as a function of postnatal age. Neither high- nor low-affinity calcium dyes demonstrated differences in peak NMDA-induced [Ca(2+)](i) increases between neurons from younger and older animals. However, neurons from older animals were uniformly distinguished from those from younger animals by their subsequent loss of [Ca(2+)](i) homeostasis. Because of the role of mitochondrial Ca(2+) buffering in [Ca(2+)](i) homeostasis, we measured NMDA-induced changes in mitochondrial membrane potential (DeltaPsi) as a function of postnatal age. NMDA markedly dissipated DeltaPsi in neurons from mature rats, but minimally in those from younger rats. These data demonstrate that, in cultures of postnatal hippocampal neurons, (a) vulnerability to excitotoxicity increases as a function of the postnatal age of the animal from which they were harvested, and (b) developmental regulation of vulnerability to NMDA occurs at the level of the mitochondrion.

Conotoxin-sensitive and conotoxin-resistant Ca2+ currents in fish retinal ganglion cells.

Using whole-cell patch-clamp methods, we tested whether omega-toxins from Conus block voltage-gated Ca2+ currents in teleost central neurons. The fractions omega-CTx-GVIA and omega-CTx-MVIIC, together with omega-toxins from Agelenopsis, the dihydropyridine BAY-K-8644, and voltage steps, produced effects indicating three types of Ca2+ current in dissociated goldfish retinal ganglion cells. One was activated by depolarization of most cells beyond -65 mV, primed at -95 mV but not at -45 mV, reduced by Ni2+, and unchanged by conotoxins, agatoxins, or BAY-K-8644. The second type constituted more than three-quarters of the total Ca2+ current in all cells, and at test potentials more positive than -30 mV, was reduced consistently by omega-CTx-GVIA, omega-CTx-MVIIC, and omega-Aga-IA, but not omega-Aga-IVA. The third Ca2+ current type was augmented by BAY-K-8644 at test potentials as negative as -45 mV, even in the presence of omega-CTx-GVIA. Replacement of extracellular Ca2+ by Ba2+ augmented current amplitude and slowed current decay. Conditioning depolarizations reduced Ca2+ current amplitude less than did omega-CTx-GVIA, and slowed current decay to imperceptible rates. These results provide the first description of conotoxin-sensitive, voltage-gated Ca2+ current recorded from teleost central neurons. Although most of the high-threshold Ca2+ current in these cells is blocked by omega-CTx-GVIA, it is also Ni(2+)-sensitive, and relatively resistant to omega-Aga-IIIA. The voltage sensitivities of low-and high-threshold Ca2+ current may suit current recruitment in situ after light-evoked hyperpolarizations end, and after light-evoked depolarizations begin, respectively.

(-)-baclofen and gamma-aminobutyric acid inhibit calcium currents in isolated retinal ganglion cells.

Of the various synaptic inputs known to converge upon retinal ganglion cells, the major inhibitory inputs are thought to be GABAergic. Although gamma-aminobutyric acid (GABA) is known to activate anion-selective ion channels in retinal ganglion cells, we have tested the possibility that GABA can also modulate cationic conductances in these cells, as seen in other central and peripheral neurons. Specifically, we have made whole-cell patch-clamp recordings to test whether voltage-gated calcium currents in isolated goldfish retinal ganglion cells are sensitive to GABAB receptor ligands. (-)-Baclofen and GABA inhibited calcium currents activated by moderately long depolarizations and, during large depolarizations (e.g., to 0 mV), also appeared to accelerate the rate of current decay. The calcium current inhibition induced by (-)-baclofen and GABA was not prevented by 2-hydroxysaclofen, phaclofen, or bicuculline, even though bicuculline suppressed a GABA-activated conductance in these cells. These results demonstrate the presence of baclofen- and GABA-sensitive calcium currents in vertebrate retinal ganglion cells as well as the coexistence of GABAA and GABAB receptors in individual retinal ganglion cells.

omega-Aga-I: a presynaptic calcium channel antagonist from venom of the funnel web spider, Agelenopsis aperta.

Spider venoms are proving to be important sources of specific ion channel toxins. Venom of Agelenopsis aperta, a funnel web spider, contains a class of polypeptide toxins which blocks neuromuscular synapses at nanomolar concentrations. Detailed physiological analyses of block caused by one of these toxins, omega-Aga-I, show that it suppresses transmitter release at insect and frog neuromuscular junctions and blocks calcium spikes in insect neuronal cell bodies. omega-Aga-I may define a binding site on neuronal calcium channels which is common to both vertebrates and invertebrates.

Excitotoxic degeneration is initiated at non-random sites in cultured rat cerebellar neurons.

Prolonged stimulation of cultured cerebellar neurons by kainic acid (KA) leads to death of neurons first evident from the swelling of soma and neurites. Stimulation is accompanied by increases in [Ca2+]i and [Na+]i as monitored using digital imaging microfluorimetry. "Blebs" tended to form on neurites with the highest increases in [Ca2+]i. Points of Ca2+ entry into neurites via glutamate-receptor-gated channels predicted where approximately 80% of blebs would form tens of minutes later. These sites were close to neurite intersections where there was a high likelihood of synaptic contacts and were enriched in mitochondria as revealed by rhodamine 123 staining. Ca2+, but not Na+ entry, produced a loss of mitochondrial potential. Prolonged KA, but not 50K, applications could fully dissipate the neuronal Na+ gradient. Recovery of resting [Na+]i was delayed by Ca2+ loading. We propose that blebs form at certain synaptic regions due to localized ionic fluxes and local Ca2+ overloading. Increased [Ca2+]i may hamper restoration of normal [Na+]i permitting local osmotic swelling as well as activation of Ca(2+)-dependent enzymes and other processes. Na+ may slow, block, or reverse Na/Ca exchange and enhance swelling. These conditions could not be reproduced by global changes in ion concentrations produced by Ca2+ or Na+ ionophores. The earliest stages of excitotoxicity thus appear to be manifestations of localized disruptions of ionic homeostasis mediated by Ca2+ overload and Na+ influx.

Na(+)-Ca2+ exchanger-like immunoreactivity and regulation of intracellular Ca2+ levels in fish retinal ganglion cells.

1. We have used two experimental approaches to examine regulation of intracellular calcium ion levels in fish retinal ganglion cells. In the first set of experiments, we ratio-imaged fura-2 emission intensity to estimate the concentration of free intracellular calcium ions ([Ca2+]i) in isolated goldfish retinal ganglion cells depolarized by increases in extracellular levels of potassium ions ([K+]o), in the presence and absence of extracellular sodium ions (Na+). Stepwise increases in [K+]o from 5 mM to as high as 60 mM produced stepwise increases in [Ca2+]i. These increases were sustained in the absence of external Na+, but transient and smaller in the presence of external Na+. The decline of [Ca2+]i in high-K, Na(+)-containing saline could be reversed by application of the ionophore monensin, or by replacement of external Na+ with either N-methyl-D-glucamine or lithium. In Na(+)-containing saline, [Ca2+]i fell to control levels after [K+]o was restored to control levels. 2. In the second set of experiments, we assessed Na(+)-Ca2+ exchanger-like immunoreactivity in goldfish retinal ganglion cells with the use of a polyclonal antiserum directed against Na(+)-Ca2+,K+ exchanger purified from bovine rod outer segments. This antiserum specifically stained the somata, neurites, and growth cones of isolated ganglion cells, the outer segments of rod photoreceptors, and (on Western blots prepared from mechanically isolated rods) protein displaying an apparent molecular mass of 210 kDa. 3. These results provide measurements of changes in [Ca2+]i of retinal ganglion cells depolarized in Na(+)-containing saline, and the distribution and apparent molecular weight of Na(+)-Ca2+ exchanger-like immunoreactivity in teleost retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure scheme separates effects of electric shock and electric field for honey bees, Apis mellifera L.

Mechanisms to explain disturbance of honey bee colonies under a 765-kV, 60-Hz transmission line [electric (E) field = 7 kV/m] fall into two categories: direct bee perception of enhanced in-hive E fields, and perception of shock from induced currents. The same adverse biological effects previously observed in honey bee colonies exposed under a 765-kV transmission line can be reproduced by exposing worker bees to shock or E field within elongated hive entranceways (= tunnels). Exposure to intense E field caused disturbance only if bees were in contact with a conductive substrate. E-field and shock exposure can be separated and precisely defined within tunnels, eliminating dosimetric vagaries that occur when entire hives are exposed to E field.

Mechanism of biological effects observed in honey bees (Apis mellifera, L.) hived under extra-high-voltage transmission lines: implications derived from bee exposure to simulated intense electric fields and shocks.

This work explores mechanisms for disturbance of honey bee colonies under a 765 kV, 60-Hz transmission line [electric (E) field = 7 kV/m] observed in previous studies. Proposed mechanisms fell into two categories: direct bee perception of enhanced in-hive E fields and perception of shock from induced currents. The adverse biological effects could be reproduced in simulations where only the worker bees were exposed to shock or to E field in elongated hive entranceways (= tunnels). We now report the results of full-scale experiments using the tunnel exposure scheme, which assesses the contribution of shock and intense E field to colony disturbance. Exposure of worker bees (1,400 h) to 60-Hz E fields including 100 kV/m under moisture-free conditions within a nonconductive tunnel causes no deleterious affect on colony behavior. Exposure of bees in conductive (e.g., wet) tunnels produces bee disturbance, increased mortality, abnormal propolization, and possible impairment of colony growth. We propose that this substrate dependence of bee disturbance is the result of perception of shock from coupled body currents and enhanced current densities postulated to exist in the legs and thorax of bees on conductors. Similarly, disturbance occurs when bees are exposed to step-potential-induced currents. At 275-350 nA single bees are disturbed; at 600 nA bees begin abnormal propolization behavior; and stinging occurs at 900 nA. We conclude that biological effects seen in bee colonies under a transmission line are primarily the result of electric shock from induced hive currents. This evaluation is based on the limited effects of E-field exposure in tunnels, the observed disturbance thresholds caused by shocks in tunnels, and the ability of hives exposed under a transmission line to source currents 100-1,000 times the shock thresholds.

Laboratory investigations of the electrical characteristics of honey bees and their exposure to intense electric fields.

Bees exposed to 60-Hz electric (E) fields greater than 150 kV/m show field-induced vibrations of wings, antennae, and body hairs. They also show altered behavior if exposed while in contact with a conductive substrate. Measurements indicate that approximately 240 nA is coupled to a bee standing on a conductive substrate in a 100-kV/m E field. In lab experiments, bee disturbance and sting result from exposure to E field greater than 200 kV/m (bee current greater than 480 nA) and reduced voluntary movements at greater than 300 kV/m (greater than 720 nA bee current) only if the bee is on a conductive substrate. It is hypothesized that in the latter situation coupled bee current drains through the lower thorax and legs to the conductive substrate, and that the resulting enhanced current density in these regions is the cause of observed responses. The observation that bees exposed to intense E fields on an insulator show vibration of body parts but no behavioral response suggests that vibration contributes little to the disturbance of bees in intense E fields. Lab measurements of bee impedance from front-to-rear leg pairs were made on wet and dry conductors. Measurements validate the selection of 1 M omega as a middle value for bee impedance used in the design of devices used to generate step-potential-induced currents in bees.

Biological effects of a 765-kV transmission line: exposures and thresholds in honeybee colonies.

Honeybee colonies exposed under a 765-kV, 60-Hz transmission line at 7 kV/m show the following sequence of effects: 1) increased motor activity with transient increase in hive temperature; 2) abnormal propolization; 3) impaired hive weight gain; 4) queen loss and abnormal production of queen cells; 5) decreased sealed brood; and 6) poor winter survival. When colonies were exposed at 5 different E fields (7, 5.5, 4.1, 1.8, and 0.65-0.85 kV/m) at incremental distances from the line, different thresholds for biologic effects were obtained. Hive net weights showed significant dose-related lags at the following exposures: 7 kV/m, one week; 5.5 kV/m, 2 weeks; and 4.1 kV/m, 11 weeks. The two lowest exposure groups had normal weight after 25 weeks. Abnormal propolization of hive entrances did not occur below 4.1 kV/m. Queen loss occurred in 6 of 7 colonies at 7 kV/m and 1 of 7 at 5.5 kV/m, but not below. Foraging rates were significantly lower only at 7 and 5.5 kV/m. Hive weight impairment and abnormal propolization occur at lower E-field intensity than other effects and limit the "biological effects corridor" of the transmission line to approximately 23 m beyond a ground line projection of each outer phase wire. Intrahive E fields of 15-100 kV/m were measured with a displacement current sensor. Step-potential-induced currents up to 0.5 microA were measured in an electrically equivalent bee model placed on the honeycomb in a hive exposed at 7 kV/m. At 1.8 kV/m body currents were a few nanoamperes, or two orders of magnitude lower, and these colonies showed no effects. E-field versus electric shock mechanisms are discussed.

Calcium ion levels in resting and depolarized goldfish retinal ganglion cell somata and growth cones.

1. We have estimated free, intracellular calcium ion concentrations ([Ca]i) in isolated retinal ganglion cells of adult goldfish by ratio-imaging fura-2 emission intensity at two excitation wavelengths. Here we describe [Ca]i in these cells, both at rest and during depolarization by elevated levels of extracellular potassium ions ([K]o). 2. [K]o was varied between 5 and 60 mM in sodium-free, tetrodotoxin-containing salines. Ganglion cell membrane potential, measured with patch electrodes, fell with each increment of [K]o used, from approximately -70 mV in 5 mM K+ to approximately -20 mV in 60 mM K+. 3. In control saline, [Ca]i was roughly 120 nM in cell somata and at least twofold higher in their growth cones. [Ca]i increased in both somata and growth cones to as high as 1.5 microM in salines containing 60 mM K+. [Ca]i exceeded 1.5 microM in some cells in high-K+ salines, although these levels could not be quantified accurately with fura-2. 4. Increases in [Ca]i elicited by elevated [K]o persisted for the duration of the exposure to high-K+ saline and were blocked by replacement of most of the bath Ca2+ by Co2+. These increases in [Ca]i were also sensitive to dihydropyridine calcium-channel ligands, viz., enhanced by BAY K 8644 (3 microM) and antagonized by nifedipine (10 microM). 5. Partial recovery of control [Ca]i occurred when [K]o was reduced to 5 mM after exposure to high-K+ saline and in high-K+ saline when nifedipine was included. These results show that goldfish retinal ganglion cells can partially buffer intracellular Ca2+ in the absence of extracellular Na+ ions. 6. These results provide measurements of the changes in [Ca]i brought about by depolarization of goldfish retinal ganglion cells in Na(+)-free salines. In these salines, at least part of the increase in [Ca]i appears to result from Ca2+ influx through a voltage-activated, noninactivating calcium conductance in the somata and growth cones of these cells. These measurements complement whole-cell patch-clamp and vibrating microprobe recordings from the somata and neurites of these cells and also immunocytochemical studies and patch-clamp measurements in amphibian, reptilian, and mammalian retinal ganglion cells.

Differential antagonism of transmitter release by subtypes of omega-agatoxins.

1. The omega-agatoxins from Agelenopsis aperta spider venom are a diverse group of voltage-sensitive calcium channel antagonists. Subtypes of omega-agatoxins are distinguished as type I (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IC), type II (omega-Aga-IIA and omega-Aga-IIB), and type III (omega-Aga-IIIA and omega-Aga-IIIB). All except type III toxins block calcium channels in insect motor nerve terminals and in neuronal cell bodies at nanomolar concentrations. 2. The potency and maximum level of block of the excitatory junctional potential (EJP) by omega-agatoxins are dependent on the extracellular calcium concentration ([Ca]o). Saturating concentrations of type I or II omega-agatoxins block 97-99% of the evoked EJP in low [Ca]o (0.75 mM) saline. A remnant of the EJP (1-3%) that persists after toxin exposure suggests that a small amount of voltage-dependent calcium entry is toxin resistant. When [Ca]o is elevated to 5 mM, this resistant component increases dramatically, revealing differences between type I and type II block. Under these conditions, 60-70% of the EJP is resistant to type I toxins and approximately 20% is resistant to type II toxins. 3. Sequential application of type I and II toxins in high [Ca]o leads to enhanced block of the EJP, suggesting that type I and II omega-agatoxins may block calcium channels by different mechanisms. 4. Type I and type II omega-agatoxins also block calcium channels in the somata of locust dorsal unpaired median (DUM) neurons. In agreement with studies on neuromuscular transmission, block of barium action potentials is incomplete after either type I or type II toxin exposure and combined application of the toxins results in enhanced block. 5. Partial calcium channel antagonism by type I and type II toxins could be explained either by altered kinetics of toxin-modified channels or by selectivity for different subtypes of presynaptic calcium channels.

Neuropeptide Y and pancreatic polypeptide reduce calcium currents in acutely dissociated neurons from adult rat superior cervical ganglia.

We examined the effects of neuropeptide Y (NPY) and pancreatic polypeptide on calcium currents (ICa) in acutely dissociated neurons from the adult rat superior cervical ganglion. We found that NPY inhibited the ICa with an estimated IC50 value of 140 nM. This inhibitory effect appeared to be restricted to a subset of cells which were smaller in diameter than the general population. The effect of NPY on the ICa was prevented by pretreatment with pertussis toxin, suggesting the involvement of a GTP-binding protein of the Gi or Go subtype. omega-conotoxin GVIA also occluded the effects of NPY, which suggests that these were directed toward N-type Ca++ channels. The effects of NPY were mimicked by the fragment NPY (13-36) but not by peptide YY, indicating that a receptor distinct from a Y1- or a Y2-like NPY receptor was involved. Finally, we also observed that pancreatic polypeptide inhibited the ICa, suggesting that a pancreatic polypeptide receptor is also present on superior cervical ganglion neurons.

Mechanism of presynaptic inhibition by neuropeptide Y at sympathetic nerve terminals.

Calcium influx through voltage-sensitive Ca2+ channels is the normal physiological stimulus for the activity-dependent release of neurotransmitters at synaptic contacts. It has been postulated that presynaptic inhibition of transmitter release is due to a reduction in Ca2+ influx at the nerve terminal, which could result from the direct inhibition of Ca2+ channels. Neuropeptide Y and noradrenaline act as cotransmitters at many sympathetic synapses. Both of these substances produce presynaptic inhibition and can inhibit Ca2+ currents in the soma of sympathetic neurons. Here we provide direct evidence that presynaptic inhibition produced by neuropeptide Y at sympathetic nerve terminals is associated with a reduction in Ca2+ influx and that this is due to the selective inhibition of neuronal N-type Ca2+ channels.

Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine.

Digital-imaging microfluorimetry of the oxidation of hydroethidine (HEt) to ethidium can be used to monitor superoxide (O2-) production selectively within individual rat hippocampal pyramidal neurons in culture and in brain slices. Under assay conditions, oxidation was not accomplished by hydroxyl radical, singlet O2, H2O2, or nitrogen radicals. Neuronal O2- production varied with metabolic activity and age. O2- generation increased after treatment with AMPA, kainic acid, and NMDA, and the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenyl hydrazone, but usually not after depolarization (50 mM K+). O2- concentrations were sensitive to scavengers and nitric oxide. HEt oxidation was higher in Ca(2+)-containing versus Ca(2+)-free saline. However, Ca2+ ionophores did not increase oxidation greatly. H2O2 application produced a secondary increase in O2-. The major source of O2- under basal and stimulated conditions appeared to be the mitochondria. Consistent with this, ethidium staining in dendrites was punctate, colocalized with mitochondria, and blocked by CN-.

Changes in mitochondrial function resulting from synaptic activity in the rat hippocampal slice.

Digital imaging microfluorimetry was used to visualize changes in mitochondrial potential and intracellular Ca2+ concentration, [Ca2+]i, in thick slices of rat hippocampus. Electrical activity, especially stimulus train-induced bursting (STIB) activity, produced slow, prolonged changes in mitochondrial potential within hippocampal slices as revealed by fluorescence measurements with rhodamine dyes. Changes in mitochondrial potential showed both temporal and spatial correlations with the intensity of the electrical activity. Patterned changes in mitochondrial potential were observed to last from tens of seconds to minutes as the consequence of epileptiform discharges. STIB-associated elevations in [Ca2+]i were also prolonged and exhibited a spatial pattern similar to that of the mitochondrial depolarization. The mitochondrial depolarization was sensitive to TTX and glutamate receptor blockers ([Mg2+]o and CNQX or DNQX plus D-AP-5) and to the inhibition of glutamate release by activation of presynaptic NPY receptors. The monitoring of mitochondrial potential in slice preparations provides a new tool for mapping synaptic activity in the brain and for determining the roles of mitochondria in regulation of brain synaptic activity.

Omega-agatoxins: novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis aperta) venom.

A new series of polypeptide presynaptic antagonists ("omega-agatoxins") was purified from venom of the funnel web spider Agelenopsis aperta. Physiological data indicate that all of these peptides are antagonists of voltage-sensitive calcium channels. Although all three omega-agatoxins (Aga) described here (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IIA) block insect neuromuscular transmission presynaptically, biochemical data permit their subclassification as Type I and Type II toxins. Type I toxins (omega-Aga-IA and -IB) are 7.5 kDa, have closely related amino acid sequences, and exhibit characteristic tryptophan-like UV absorbance spectra. Complete Edman sequencing of omega-Aga-IA reveals it to be a 66-amino acid polypeptide containing 9 cysteines and 5 tryptophan residues. omega-Aga-IIA, a Type II toxin, is 11 kDa, shows limited amino acid sequence similarity to the Type I toxins, and exhibits mixed tryptophan- and tyrosine-like absorbance. Nanomolar concentrations of omega-Aga-IIA inhibit the specific binding of 125I-labeled omega-conotoxin GVIA to chick synaptosomal membranes while omega-Aga-IA and -IB have no effect under identical conditions. The omega-agatoxins thus are defined as two subtypes of neuronal calcium channel toxins with different structural characteristics and calcium channel binding specificities.