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

1. Brief synaptic stimulation, or exposure to Conus textile venom (CtVm), t riggers an afterdischarge in the bag cell neurones of Aplysia. This is asso ciated with an elevation of intracellular calcium ([Ca2+](i)) through Ca2release from intracellular stores and Ca2+ entry through voltage-gated Ca2 channels and a non-selective cation channel. The afterdischarge is followe d by a prolonged (similar to 18 h) refractory period during which the abili ty of both electrical stimulation and CtVm to trigger afterdischarges or el evate [Ca2+](i) is severely attenuated. By measuring the response of isolat ed cells to CtVm, we have now tested the contribution of different sources of Ca2+ elevation to the onset of the prolonged refractory period. 2. CtVm induced an increase in [Ca2+](i) in both normal and Ca2+-free salin e, in part by liberating Ca2+ from a store sensitive to thapsigargin or cyc lopiazonic acid, but not sensitive to heparin. 3. In the presence of extracellular Ca2+, the neurones became refractory to CtVm after a single application but recovered following similar to 24 h, w hen CtVm could again elevate [Ca2+](i). However, this refractoriness did no t develop if CtVm was applied in Ca2+-free saline. Thus, elevation of [Ca2](i) alone does not induce refractoriness to CtVm-induced [Ca2+](i) elevati on, but Ca2+ influx triggers this refractory-like state. 4. CtVm produces a depolarization of isolated bag cell neurones. To determi ne if Ca2+ influx through voltage-gated Ca2+ channels, activated during thi s depolarization, caused refractoriness to CtVm-induced [Ca2+](i) elevation , cells were depolarized with high external potassium (60 mM), which produc ed a large increase in [Ca2+](i). Nevertheless, subsequent exposure of the cells to CtVm produced a normal response, suggesting that Ca2+ influx throu gh voltage-gated Ca2+ channels does not induce refractoriness. 5. As a second test for the role of voltage-gated Ca2+ channels, these chan nels were blocked with nifedipine. This drug failed to prevent the onset of refractoriness to CtVm-induced [Ca2+](i) elevation, providing further evid ence that Ca2+ entry through voltage-gated Ca2+ channels does not initiate refractoriness. 6. To examine if Ca2+ entry through the CtVm-activated, non-selective catio n channel caused refractoriness, neurones were treated with a high concentr ation of TTX, which blocks the cation channel. TTX protected the neurones f rom the refractoriness to [Ca2+](i) elevation produced by CtVm in Ca2+-cont aining medium. 7. Using clusters of bag cell neurones in intact abdominal ganglia, we comp ared the ability of nifedipine and TTX to protect the cells from refractori ness 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 prese nce of nifedipine resulted in refractoriness. 8. Our data indicate that Ca2+ influx through the non-selective cation chan nel renders cultured bag cell neurones refractory to repeated stimulation w ith CtVm. Moreover, the refractory period of the afterdischarge itself may also be initiated by Ca2+ entry through this cation channel.