Voltage-dependent activation of potassium current in Helix neurones by endogenous cellular calcium

Abstract

1. The effect of endogenous Ca on potential-dependent K current IKD, was examined in identifiable neurones of Helix aspersa. The suction pipette method of internal perfusion was used along with a combined voltage-clamp method in which the membrane potential was measured by a separate glass micro-electrode and the current was passed by the suction pipette. Activation of the potential-dependent A current, IA, was prevented by using holding potentials of -40 mV where IA is inactivated and by the addition of the A-current blocker 4-aminopyridine. Activation of K currents by transmembrane Ca current, IKCa, was suppressed by Co substitution for Ca ion extracellularly. 2. Under these conditions, IKD rose to a peak value and then subsided to a steady level. The current-voltage (I-V) relationship for peak IKD had an upward bump at about +50 mV that gave it an S-shape. The I-V curve for steady IKD rose continuously. Peak and steady IKD were reduced by perfusing with EGTA or F ions intracellularly. The EGTA effect occurred at intracellular Ca activity levels below 10(-7) M. Increases in the concentration of EGTAi at constant Cai had no additional effect; however, recovery experiments do not allow us to rule out some direct action of EGTA on IKD. 3. Prolonged extracellular perfusion with Co-substituted solutions also reduced IKD and the effects occurred more quickly when the solutions were made hypertonic or caffeine was added to them. The peak transient was abolished, and the small remaining steady IKD (about 5-10% of normal peak IKD) was blocked by tetraethylammonium. IKD could be restored by the temporary reintroduction of Ca in the extracellular solution. 4. The S-shape of the peak I-V relationship for IKD may be due to Ca released from an endogenous site by membrane depolarization. The reduction of steady and peak IKD to very low values by Ca chelators or prolonged perfusion with Ca-free solutions indicates that Cai is important for activation of these K channels. 5. Three cellular structures were identified in electron micrographs of freeze-fractured neurones that could be involved in potential-dependent endogenous Ca release. These were a restricted extracellularly space, an intracellular membrane system of endoplasmic reticulum that may be fused to the internal face of the plasma membrane (the subsurface cisterns of Henkart & Nelson, 1979), and intracellular vesicles that also may be fused to the plasma membrane.