Activation of a Ca2+-permeable cation channel produces a prolonged attenuation of intracellular Ca2+ release in Aplysia bag cell neurones

Brief synaptic stimulation, or exposure to Conus textile venom (CtVm), triggers an afterdischarge in the bag cell neurones of Aplysia. This is associated with an elevation of intracellular calcium ([Ca2+]i) through Ca2+ release from intracellular stores and Ca2+ entry through voltage-gated Ca2+ channels and a non-selective cation channel. The afterdischarge is followed by a prolonged (∼18 h) refractory period during which the ability of both electrical stimulation and CtVm to trigger afterdischarges or elevate [Ca2+]i is severely attenuated. By measuring the response of isolated cells to CtVm, we have now tested the contribution of different sources of Ca2+ elevation to the onset of the prolonged refractory period. CtVm induced an increase in [Ca2+]i in both normal and Ca2+-free saline, in part by liberating Ca2+ from a store sensitive to thapsigargin or cyclopiazonic acid, but not sensitive to heparin. In the presence of extracellular Ca2+, the neurones became refractory to CtVm after a single application but recovered following ∼24 h, when CtVm could again elevate [Ca2+]i. However, this refractoriness did not develop if CtVm was applied in Ca2+-free saline. Thus, elevation of [Ca2+]i alone does not induce refractoriness to CtVm-induced [Ca2+]i elevation, but Ca2+ influx triggers this refractory-like state. CtVm produces a depolarization of isolated bag cell neurones. To determine if Ca2+ influx through voltage-gated Ca2+ channels, activated during this depolarization, caused refractoriness to CtVm-induced [Ca2+]i elevation, cells were depolarized with high external potassium (60 mm), which produced a large increase in [Ca2+]i. Nevertheless, subsequent exposure of the cells to CtVm produced a normal response, suggesting that Ca2+ influx through voltage-gated Ca2+ channels does not induce refractoriness. As a second test for the role of voltage-gated Ca2+ channels, these channels were blocked with nifedipine. This drug failed to prevent the onset of refractoriness to CtVm-induced [Ca2+]i elevation, providing further evidence that Ca2+ entry through voltage-gated Ca2+ channels does not initiate refractoriness. To examine if Ca2+ entry through the CtVm-activated, non-selective cation channel caused refractoriness, neurones were treated with a high concentration of TTX, which blocks the cation channel. TTX protected the neurones from the refractoriness to [Ca2+]i elevation produced by CtVm in Ca2+-containing medium. Using clusters of bag cell neurones in intact abdominal ganglia, we compared the ability of nifedipine and TTX to protect the cells from refractoriness to electrical stimulation. Normal, long-lasting afterdischarges could be triggered by stimulation of an afferent input after a period of exposure to CtVm in the presence of TTX. In contrast, exposure to CtVm in the presence of nifedipine resulted in refractoriness. Our data indicate that Ca2+ influx through the non-selective cation channel renders cultured bag cell neurones refractory to repeated stimulation with CtVm. Moreover, the refractory period of the afterdischarge itself may also be initiated by Ca2+ entry through this cation channel. Transient changes in [Ca2+]i produce short- or long-term alterations in ion channels, secretion and gene expression (Partridge & Swandulla, 1988; Latorre et al. 1989; Sobel & Tank, 1994; Clapham, 1995; Ghosh & Greenberg, 1995; Scott et al. 1995; Simpson et al. 1995; Levitan & Kaczmarek, 1997). However, the ability of an elevation in Ca2+ to produce a specific effect depends on the location of the source of Ca2+. For example, the release of classical neurotransmitters is triggered by Ca2+ entry through voltage-gated Ca2+ channels at active zones, which produce a microdomain of high Ca2+ near the mouth of the channels where synaptic vesicles are docked (Stanley, 1997). Recent work has also suggested that changes in gene transcription produced by elevation of [Ca2+]i do not depend on global Ca2+, but can only be induced by Ca2+ entry through specific subtypes of voltage-gated Ca2+ channels, perhaps because the Ca2+-sensitive signalling pathways that translocate to the nucleus are physically coupled to these channels (Deisseroth et al. 1998). The bag cell neurones of Aplysia are a model system used to study the regulation of prolonged changes in excitability and [Ca2+]i. These neurones, located in the abdominal ganglion in two symmetrical clusters of 200–400 neurones, control a sequence of reproductive behaviours culminating in egg-laying behaviour (Kupfermann, 1967; Kupfermann & Kandel, 1970; Pinsker & Dudek, 1977; Conn & Kaczmarek, 1989). In response to brief stimulation, the normally silent bag cell neurones undergo an ∼30 min afterdischarge, consisting of prolonged depolarization and action potential firing, during which egg-laying hormone and a number of other neuropeptides are secreted (Rothman et al. 1983; Conn & Kaczmarek, 1989). Following an afterdischarge, the bag cells enter an ∼18 h refractory period, during which another lengthy afterdischarge cannot be elicited. This prolonged refractory period is believed to prevent the reinitiation of the sequence of reproductive behaviours. Prior work suggests that an elevation of [Ca2+]i during the afterdischarge triggers the prolonged refractory period (Kaczmarek & Kauer, 1983). During an afterdischarge, Ca2+ is released from intracellular stores (Fisher et al. 1994), and there is an enhancement of Ca2+ entry through plasma membrane channels, including voltage-dependent Ca2+ channels (Strong et al. 1987) and a non-selective cation channel that appears to be responsible for the maintained depolarization of the cells (Wilson et al. 1996). If Ca2+ is omitted from the external medium, multiple afterdischarges can be evoked, although these are generally much shorter in duration. Moreover, a refractory-like state can be induced artificially by treating the cells with a Ca2+ ionophore (Kaczmarek & Kauer, 1983). However, it is not known whether a specific source of Ca2+ normally contributes to the prolonged refractory period. The neurotransmitter that triggers afterdischarges is not known. In the isolated CNS, an afterdischarge can be induced in bag cell neurones by stimulating one of the pleuroabdominal connectives, which contain the axons of these neurones. Afterdischarges that appear identical in all aspects to those evoked by electrical stimulation can be induced by venom from the molluscivorous marine snail Conus textile (Wilson et al. 1996). Venom from the snails of the Conus genus has proven extremely useful in the study of ion channels, yielding toxins that act on, for example, voltage-dependent Na+ and Ca2+ channels (Olivera et al. 1990). In cultured bag cell neurones, Conus textile venom (CtVm) depolarizes the cells by activating a slow, voltage-dependent, Ca2+-permeable, non-selective cation channel (which can be blocked by TTX). When applied to the intact cluster, CtVm elicits an afterdischarge that is followed by the normal prolonged refractory period (Wilson et al. 1996). Using isolated bag cell neurones, we now show that CtVm triggers both Ca2+ entry and the release of Ca2+ from intracellular stores. We also demonstrate that this CtVm-induced [Ca2+]i elevation becomes refractory, for ∼24 h, following a single application. This refractory-like state is produced by Ca2+ entry through the TTX-sensitive, non-selective cation channel, while other pathways of Ca2+ elevation, including voltage-dependent Ca2+ channels and intracellular Ca2+ stores, fail to elicit this refractoriness. In addition, block of this cation channel prevents the onset of true refractoriness to afterdischarge in intact clusters in response to CtVm. Our findings suggest that the non-selective cation channel, that drives afterdischarges, is selectively located in close physical proximity to a Ca2+-sensitive mechanism that produces a very long lasting change in the excitability of the bag cell neurones.