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

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