Knox, R.J., Jonas, E.A., Kao, L.S., Smith, P.J., Connor, J.A. and Kaczmarek, L.K.
Ca2+ influx and activation of a cation current are coupled to intracellular Ca2+ release in peptidergic neurons of Aplysia californica
Journal of Physiology, 494, (3), . (PMID:1316764).
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1. Stimulation of inputs to bag cell neurons in the abdominal ganglion of Aplysia californica causes an increase in their intracellular Ca2+ concentration ([Ca2+]i). We have used thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ pump, to analyse the effects of Ca2+ released from intracellular stores on the electrophysiological responses of bag cell neurons.
2. Using digital imaging of fura-2-loaded isolated bag cell neurons we found that thapsigargin rapidly evoked an increase in [Ca2+]i in somata, with smaller increases in neurites. Thapsigargin-induced elevation of [Ca2+]i peaked at about 1 microM within 5-10 min and then decayed to basal levels by 30 min.
3. Placement of an extracellular vibrating Ca(2+)-selective microelectrode to within 1 micron of somata revealed a relatively large steady-state Ca2+ efflux. Thapsigargin produced a rapid increase in Ca2+ influx. Changes in Ca2+ flux were not detected at neurites.
4. Thapsigargin produced a small depolarization in isolated bag cell neurons in artificial sea water (ASW). Sometimes enhanced depolarizations were observed when extracellular Na+ was replaced by TEA or Tris, but not N-methyl-D-glucamine (NMDG). The depolarization was not blocked by 100 microM tetrodotoxin (TTX), removal of extracellular Ca2+ (0.5 mM EGTA) or addition of 10 mM Co2+ to the bath solution.
5. In voltage-clamp experiments, thapsigargin induced an inward current (ITg) that was recorded in Ca(2+)-free media containing TEA or Tris substituted for Na+. The apparent reversal potential of ITg was -16.8 +/- 1.2 mV in TEA-ASW. Induction of ITg was inhibited in neurons that were microinjected with the Ca2+ chelator BAPTA-Dextran70 or treated with the membrane-permeant analogue BAPTA AM. Activation of ITg was not observed when Na+ was replaced with NMDG. Manipulation of [Na+]o and [K+]o produced shifts in the reversal potential of ITg consistent with the underlying channels being permeable to both Na+ and K+.
6. Thapsigargin did not alter the amplitude or kinetics of voltage-activated Ba2+ currents, but in some experiments it did increase the amplitude of a component of outward K+ current.
7. Thapsigargin neither induced bag cell neurons within the intact ganglion to depolarize and fire spontaneously, nor did it alter the frequency or duration of firing of an electrically stimulated bag cell after-discharge.
8. We conclude that thapsigargin-sensitive Ca2+ pools are present predominantly in the somata of bag cell neurons. Ca2+ that is released from thapsigargin-sensitive Ca2+ stores activates a non-selective cation current that may help sustain depolarization of the somata, but does not by itself trigger an after-discharge.
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