Calcium current‐dependent and voltage‐dependent inactivation of calcium channels in Helix aspersa

Abstract

Inactivation of the Ca channels was examined in isolated nerve cell bodies of H. aspersa using the suction pipette method for voltage clamp and internal perfusion. Satisfactory suppression of outward currents was essential. This was achieved over most of the voltage range by substitution of Cs+ for K+ and by the use of TEA [tetraethylammonium] intra- and extracellularly and 4-AP [4-aminopyridine] extracellularly. A small time- and voltage-dependent non-specific current remained at potentials above +60 mV. In these solutions, Ca current approaches ECa [Ca conductance], but cannot be detected in the outward direction. The Ca channel appears impermeable to Cs and Tris ions. Inactivation of Ca currents occurs as a bi-exponential process. The faster rate is 10-20 times the slower rate and is .apprx. 1/20 the rate of activation. The development of inactivation during a single voltage-clamp step and the onset of inactivation produced by prepulses followed after brief intervals by a test pulse, have roughly similar time courses. The rates of inactivation increase monotonically at potentials more positive than about -25 mV. The amount of steady-state inactivation increases with membrane depolarizations to potentials of about +50 mV. At more positive potentials, steady-state inactivation is reduced. Intracellular EGTA [ethylene glycol bis(.beta.-aminoethyl ether tetraacetate)] slows the faster rate inactivation of ICa and reduces the amount of steady-state inactivation measured with a standard 2 pulse protocol. The effect is specifically related to Ca chelation and H+ are not involved. This component of inactivation is referred to as Ca current-dependent inactivation and is consistent with observations that increased Cai inactivates the Ca channel. The process does not depend upon current flow alone since Ba currents of comparable or greater magnitude have smaller initial rates of inactivation. Application of Ba2+ ion intracellularly in large concentrations has no effect on steady-state inactivation. The bi-exponential inactivation process that persists in the presence of EGTAi is similar to that occurring when extracellular Ba2+ carries current through the Ca channel. Steady-state inactivation also persists and is similar in the 2 cases. Inactivation seems voltage-dependent as well as Ca current-dependent. Diffusion models that included reasonable values for the effect of binding on diffusion, even when combined with declining influxes, did not account for this mixed form of Ca- and voltage-dependent inactivation. A compartmental model in which the particular kinetic model of voltage-dependent inactivation was not critical described the Ca current-dependent inactivation.