Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium.

Internal perfusion of Helix neurons with a solution containing potassium aspartate, MgCl2, ATP, and HEPES causes the calcium-activated potassium current (IK(Ca)) evoked by depolarizing voltage steps to decrease with time. When internal free Ca++ is strongly buffered to 10(-7) M by including 0.5 mM EGTA and 0.225 mM CaCl2 in the internal solution, IK(Ca) remains constant for up to 3 hours of perfusion. In cells where IK(Ca) is small at the start of perfusion, perfusion with the strongly buffered 10(-7) M free Ca++ solution produces increases in IK(Ca) which ultimately saturate. In cells perfused with solutions buffered to 10(-6) M free Ca++, IK(Ca) is low and does not change with perfusion. These results lead us to conclude that IK(Ca) is stable in perfused Helix neurons and that the apparent loss of IK(Ca) seen initially with perfusion is due to accumulation of cytoplasmic calcium. Since the calcium current (ICa) provides the Ca++ which activates IK(Ca) during a depolarizing pulse, ICa is also stable in perfused cells when free intracellular Ca++ is buffered. Perfusion with 1 microM calmodulin (CaM) produces no effect on IK(Ca) with either 10(-7) or 10(-6) M free internal calcium. Inhibiting endogenous CaM by including 50 microM trifluoperazine (TFP) in both the bath and the internal perfusion solution also produces no effect on IK(Ca) with 10(-7) M free internal calcium. It is concluded that CaM plays no role in IK(Ca) activation.