Regulation of cation channel voltage- and Ca2+-dependence in Aplysia bag cell neurons
Gardam, Kate Elizabeth
MetadataShow full item record
Ion channel regulation is key to the control of excitability and behaviour. In the bag cell neurons of Aplysia californica, a voltage- and Ca2+-dependent nonselective cation channel drives a ~30-minute afterdischarge, culminating in the release of egg-laying hormone. Using excised, inside-out single channel patch-clamp, this study tested the hypothesis that inositol 1,4,5-trisphosphate (IP3), which is produced during the afterdischarge, and channel-associated protein kinase C (PKC), which is activated throughout the afterdischarge, cause a left-shift (enhancement) in both the voltage- and Ca2+-dependence of the cation channel. Kinetic analysis of bag cell neuron cation channel voltage-dependence revealed that, with depolarization, the channel remained open longer and reopened more often. A cation channel subconductance was also observed, and found to be 13 pS vs. the typical 23 pS full-conductance. The cytoplasmic face of cation channel-containing patches was exposed to 1 mM ATP, as a phosphate source for channel-associated PKC, and/or 5 uM IP3. Apparent PKC-dependent phosphorylation left-shifted voltage-dependence by -3 mV, although this effect was more prominent at negative voltages (between -90 and -30 mV). Conversely, IP3 right-shifted voltage-dependence (change in V1/2 of 6 mV). Cation channel Ca2+-dependence was similar to that previously reported, with a control EC50 of 3-5 uM. This was right-shifted by PKC (EC50 = 30 uM) and even more so by IP3 (apparent EC50 = 20 M). PKC largely rescued the Ca2+ responsiveness in the presence of IP3 (EC50 = 20 uM). Unexpectedly, IP3 plus ATP resulted in an increase in channel unitary conductance at more positive voltages. The multi-faceted regulation of the bag cell neuron cation channel suggests sophisticated modulatory control. Upregulation, such as depolarization and the left-shift in voltage-dependence with PKC, would drive the afterdischarge, while counteracting effects, such as IP3 right-shifting voltage-dependence, as well as PKC and IP3 suppressing Ca2+-dependence, would simultaneously or subsequently attenuate the channel, thus preventing an interminable afterdischarge.