Unitary A-currents of rat locus coeruleus neurones grown in cell culture: rectification caused by internal Mg2+ and Na+

Abstract

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 the on-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.(ABSTRACT TRUNCATED AT 400 WORDS)