Isolation and kinetic analysis of inward currents in neuroblastoma cells

Abstract The suction pipette method for combined voltage clamp and intracellular dialysis was applied to isolate the two components of voltage-gated inward current across membranes of NIE-115 neuroblastoma cells. In order to anlayze the kinetic behavior of the Na + and Ca 2+ channels responsible for generating these components, current through K + channels was effectively blocked by substituting impermeant Cs + for internal and external K + . Block was confirmed independently by examining the effects of the application of external tetraethylammonium or Cd 2+ ; and comparing the time course of Ca 2+ tail currents with the decay of current during a maintained depolarization. Na + currents studied at 8–10°C, developed as a fourth order process giving a maximum e-fold conductance change for a 3 mV depolarization, with half activation occurring at −10 mV. The instantaneous current-voltage relationship was linear. Time constants of the activation parameter ( m ) varied from 0.5ms (−50 mV) to 3–4 ms (−10 to −40 mV) at 10°C. Inactivation ( h ) was a first order process having a time constant between 4 ms (+10 to +60mV) and 225ms (−60 mV). Steady-state inactivation for Na + channels attained a value of 0.5 at −50 mV. A slow inactivation process, however, also is involved in gating of Na + channels, and has a time course at least two orders of magnitude slower than that for h . The temperature sensitivity of Na + currents was found to be similar to that found for other preparations. Ca 2+ currents were studied at 24–29°C in the presence of internal ethyleneglycolbis-(aminoethylether)-tetra-acetate (EGTA) and an external Ca 2+ concentration of 20 mM. Ca 2+ channel activation could also be described by a fourth order process giving an e-fold conductance change for a 5–6 mV change in potential and the half activation potential of −13 mV. Internal EGTA (20 mM) did not abolish inactivation of Ca 2+ currents and no recovery from inactivation caused by a prepulse could be measured as the prepulse potential approached the null potential for Ca 2+ influx. Time constants of both activation and inactivation of Ca 2+ channels were measured between −20 and +50 mV. Currents through K + channels could be completely eliminated by substitution of K + with Cs + , although a residual non-linear leakage current remained, in addition to currents through the Na + and Ca 2+ channels. Na + channels of these mammalian neuronal cell bodies were found to have kinetics most similar to those of neurons in gastropods, and were much slower than axonal Na + channel kinetics in either mammals or poikilothermic animals, when compared at the same temperature. Measurements of the time course of both Ca 2+ channel inactivation and activation also point to a similarity between mammalian and gastropod neuronal cell bodies. Kinetics of Ca 2+ channels were found to be 20 times slower than those for Na + channels in this preparation when the latter were scaled to the same temperature. This difference explains the generation of the late depolarization phase of the action potential in these cells by Ca 2+ currents. Under the conditions described, inactivation in these cells appears to be primarily a voltagedependent process rather than one dependent on the concentration of intracellular Ca 2+ .