UNITARY A-CURRENTS OF RAT LOCUS-CERULEUS NEURONS GROWN IN CELL-CULTURE - RECTIFICATION CAUSED BY INTERNAL MG2+ AND NA+

1. We have used whole-cell and single-channel recording to study the transient outward potassium current (A-current) of rat locus coeruleus neurones grown in tissue culture. The A-current was largely inactivated at the resting potential, but could be activated from sufficiently negative holding potentials during steps positive to -50 mV. The current was sensitive to 4-aminopyridine. Another slowly activating, sustained current was similar to a delayed rectifier. 2. In theon-cell configuration the unitary conductance of channels carrying A-current was 40.9+/-2.2 pS (n = 6) with high external potassium (140 mm) and 14.8+/-1.4 pS (n = 11) with 3 mm [K+]o. The unitary current-voltage relation was not linear, but had a negative slope at very positive voltages in 3 mm [K+]o. The reversal potential changed with [K+]o as expected for a K+ channel. 3. The open state probability of A-current channels was voltage dependent, reaching a peak of 0.78+/-0.17 (seven patches). The relationships between both activation and inactivation and membrane potential were well fitted by Boltzmann expressions. Activation was half-maximum at a potential 71.9+/-11.8 mV (n = 4) positive to the resting potential (approximately -61 mV). Inactivation was half-complete 29-4+/-3.8 mV (n = 4) negative to the resting potential. There was evidence from runs analysis for slow inactivation of channels. 4. Channels showed frequent visits to substates, the most readily identifiable of which had an amplitude 0.55+/-0.04 (n = 5) of the fully open state. Other substates had amplitudes of around 0.25 and 0.75. Occupancy of substates was greater at negative membrane potentials. 5. A preliminary analysis of kinetic behaviour, treating visits to substates as openings, shows that open times are distributed as a single exponential. The open time was 16.2 ms (n = 4) at a voltage 100 mV positive to the resting potential, increasing with further depolarization. Closed times are distributed as the sum of three or four exponentials. First latency distributions are strongly voltage dependent and.show a delay, giving a sigmoidal rise to the distribution. Increasing temperature increased unitary current and reduced mean open time. 6. The mechanism of the rectification seen in the unitary current-voltage relationship was examined using excised, inside-out patches. When 140 mm-KCl solutions containing no divalent cations and no Na+ were applied to the intracellular face of such a patch. the relation between unitary A-currents and voltage became linear (slope conductance 17.8+/-1.8 ps, n = 16, when [K+]o = 3 mM). Addition of Mg2+ (2-10 mm) or Na+ (10-20 mM) to the internal surface was found to reduce outward currents, inducing rectification similar to that seen during on-cell recording. 7. Analysis of the block by intracellular Mg2+ and Na+ gave estimates for the dissociation constant for Mg2+ of 15.5+/-1.5 mM (n = 6) at zero voltage, while that for Na+ was 76.0+/-7.2 mM (n = 3). The Hill coefficient was close to 1 in each case. The voltage dependence of the block suggests an apparent valency of 0.51 for Mg2+ and 0.78 for Na+. 8. The open time was unaffected by excision of the patch from an on-cell to the inside-out configuration. Perfusion of an excised inside-out patch with Mg2+ reduced current without affecting open time, implying that the blocking ion can unbind from the closed channel. 9. Preliminary analysis of the open and closed times is consistent with a kinetic scheme with four closed, one open and two inactivated states. There must be at least two voltage-dependent transitions between states. 10. The results with intracellular blocking ions demonstrate that neuronal A-currents, like the inward rectifiers and ATP-dependent K+ channels, are modulated by internal Mg2+ and Na+. Physiologically, this phenomenon will limit the contribution of A-current to repolarization during the overshoot of the action potential. Pathologically, build up of [Na+]i during excitotoxic phenomena by reducing the effectiveness of this current may contribute to feed-forward excitation.