Calcium-mediated inactivation of the calcium conductance in caesium-loaded giant neurones of Aplysia californica

Abstract

1. The intracellular potassium in giant neurones of Aplysia californica was replaced with caesium by a method utilizing the ionophore nystatin. Because caesium ions have low permeability through potassium channels outward currents during voltage-clamp depolarization were strongly curtailed after the caesium loading procedure and the subsequent wash-out of the ionophore. 2. The calcium current elicited by a test voltage-clamp depolarization (pulse 2) was depressed following the entry of calcium elicited by a prior depolarization (pulse 1). 3. The percentage depression of the test current was a linear function of the pulse 1 current-time integral, and thus appears to be related linearly to the amount of calcium carried into the cell during pulse 1. This linear relation was maintained when calcium entry was varied by changes in external calcium concentration, by altered pulse 1 amplitude and altered pulse 1 duration. Depression was substantially reduced by injection of EGTA, and by substitution of barium for extracellular calcium. 4. The calcium current was unaffected by prior hyperpolarization of the membrane or by prior depolarizations to about ECa. Depression of the current was not altered by addition of extracellular 50 mM-TEA or by a strong hyperpolarization between the conditioning and test pulses. 5. The rate relaxation of the inward current during a given depolarization depended on the rate of entry and accumulation of free calcium. Relaxation under a given command potential became slower when calcium was partially replaced with magnesium so as to produce a smaller calcium current, or when accumulation of intracellular free calcium was retarded by injected EGTA or by barium substitution for extracellular calcium. 6 Evidence is considered that accumulation of calcium ions at the cytoplasmic surface of the membrane leads to inactivation through an action upon the calcium conductance. Reduced driving force and intracellular surface-charge neutralization do not adequately account for the observed depression of the calcium current resulting from intracellular accumulation of calcium ions.