Abstract
Ca2+-activated, non-selective cation channels feature prominently in the regulation of neuronal excitability, yet the mechanism of their Ca2+ activation is poorly defined. In the bag cell neurones of Aplysia californica, opening of a voltage-gated, non-selective cation channel initiates a long-lasting afterdischarge that induces egg-laying behaviour. The present study used single-channel recording to investigate Ca2+ activation in this cation channel. Perfusion of Ca2+ onto the cytoplasmic face of channels in excised, inside-out patches yielded a Ca2+ activation EC50 of 10 microm with a Hill coefficient of 0.66. Increasing Ca2+ from 100 nm to 10 microm caused an apparent hyperpolarizing shift in the open probability (Po) versus voltage curve. Beyond 10 microm Ca2+, additional changes in voltage dependence were not evident. Perfusion of Ba2+ onto the cytoplasmic face did not alter Po; moreover, in outside-out recordings, Po was decreased by replacing external Ca2+ with Ba2+ as a charge carrier, suggesting Ca2+ influx through the channel may provide positive feedback. The lack of Ba2+ sensitivity implicated calmodulin in Ca2+ activation. Consistent with this, the application to the cytoplasmic face of calmodulin antagonists, calmidazolium and calmodulin-binding domain, reduced Po, whereas exogenous calmodulin increased Po. Overall, the data indicated that the cation channel is activated by Ca2+ through closely associated calmodulin. Bag cell neurone intracellular Ca2+ rises markedly at the onset of the afterdischarge, which would enhance channel opening and promote bursting to elicit reproduction. Cation channels are essential to nervous system function in many organisms, and closely associated calmodulin may represent a widespread mechanism for their Ca2+ sensitivity.