ON THE SODIUM AND POTASSIUM CURRENTS OF A HUMAN NEUROBLASTOMA CELL-LINE

1. The patch-clamp method was applied to the study of ionic currents activated by depolarization of undifferentiated IMR-32 human neuroblastoma cells. Whole-cell sodium and potassium currents and single potassium ion channel currents from cell-attached patches were investigated. 2. Cells had a mean resting potential of -38 mV and mean input resistance of 1.6 G-OMEGA. Single action potentials were evoked under current clamp during the injection of depolarizing currents. 3. A voltage-dependent inward sodium current was observed which reversed at + 44 mV. A Boltzmann fit to the activation curve gave a half-maximal activation voltage of -41.6 mV and a 'slope' of 3.9 mV. The steady-state inactivation curve had a half-maximal inactivation voltage of -81 mV and a 'slope' of 9.7 mV. 4. The time-dependent activation and inactivation of the current displayed classical Hodgkin-Huxley kinetics. Values for the time constants tau-m and tau-h of 0.16 and 0.63 ms were calculated for a voltage jump from -80 to -10 mV; tau-m and tau-h decreased as the step potential was changed from -30 to +20 mV. 5. Outward currents were activated in bathing solutions substantially free of anions and could thus be attributed to potassium ions. The tail current reversed in direction on repolarization to -60 mV when the potassium concentration in the bathing solution was increased from 6 to 30 mM. When the bathing solution contained 145 mM-potassium, and the patch pipette, 95 mM, a depolarization to -10 mV from a holding potential of -60 mV evoked an inward current. 6. Outward currents were examined by using voltage pulses which depolarized the cell to -20 mV, or more positive values, from a holding potential of -80 mV and by pulses which depolarized the cell to 0 mV, or to positive values, from a holding potential of -30 mV. A Boltzmann fit of typical activation data gave a half-maximal activation voltage of 17 mV and a 'slope' of 14 mV. 7. The time course of the rising phase of the current was described by a function of the form A{1-exp [-(t-delta-t)/tau]}, where delta-t varied between 1 and 4 ms and tau varied between 4 and 27 ms, decreasing with increasing depolarization. There was no evidence for a fast transient component. 8. The amplitude of outward currents was reduced by extracellular calcium ions, cobalt ions, tetraethylammonium and 4-aminopyridine. 9. During prolonged depolarization, the time course of the current could be described by the sum of two exponentially decaying components, with time constants of around 1 and 10 s. 4-Aminopyridine selectively reduced the slower component. 10. Single-channel currents were evoked by depolarization of cell-attached patches with electrodes containing normal extracellular solution. The most frequently encountered channel had a conductance of about 20 pS. Patches usually contained either no or several such channels; the activity of a single channel, however, could be recorded during the inactivation which occurred during prolonged depolarization. 11. A detailed study was made of records obtained from one cell in response to two different voltage jumps. With the 46 mV jump, bursts of openings of 26 ms average duration occurred, separated by interburst periods of 74 ms average duration; the 'open probability' was 0.26. With the 82 mV jump, bursts could not be distinguished; the open probability was 0.90. 12. If events shorter than 1 ms were disregarded, the probability density functions for open times were single exponentials with time constants of 8.5 ms for the 46 mV jump and 18 ms for the 82 mV jump. Two exponential components were required to describe the probability density function for the closed times with time constants of 1 and 70 ms for the 46 mV jump, and 1 and 10 ms for the 82 mV jump. 13. On the basis of these probability density functions, the whole-cell outward currents would be expected to be about six times larger for a voltage jump of 82 mV than for one of 46 mV and to rise at about twice the rate. This is consistent with what was observed.