Ca2(+)‐activated K+ current involvement in neuronal function revealed by in situ single‐channel analysis in Helix neurones.

Abstract

SUMMARY1. The properties of single calcium-activated potassium channels (or C-channels) were studied in cell-attached patches using the patch-clamp technique. Experiments were performed on identified Ca2+-dependent U cells in juvenile specimens (1-2 months old) of Helix aspersa.2. The criteria used to identify C-channels were based on comparison between macroscopic C-currents and currents reconstructed from unitary recordings. Both currents had a slow activation rate at large positive potentials which turned into fast activation after large Ca2+ entries. Both currents were blocked by intracellularly injected EGTA. 4. C-channels in U cells were distributed in clusters of three to ten channels (mean 5-05 channels in seventy-five patches). Calcium channels were present in patches containing clustered C-channels. C-channels within clusters behaved independently. 5. With patch electrode containing 8 mM-calcium, C-channels opened transiently upon patch depolarization. Reopenings in quiescent depolarized patches were induced by whole-cell spikes triggered by current pulses applied to an intracellular electrode. Apparent inactivation of C-channels in depolarized patches was in fact due to a decrease in [Ca2+]i resulting from inactivation of Ca2+ channels.6. Calcium-free saline solutions in the patch electrodes prevented C-channels from opening upon patch depolarization. Entry of calcium through the surrounding membrane induced delayed openings in the patch. Peak opening probability PO occurred 330 + 30 ms after a brief Ca2+ entry with a lag period of 50-80 ms. With patch electrodes filled with Ca2+-containing saline solutions and under conditions which maximized C-channel opening, peak P1 was reached in 20-50 ms. The same value was observed for the whole-cell C-current.7. The peak P. at a given patch potential and in response to a whole-cell spike was not altered by a previous long-lasting patch depolarization, or by producing several * To whom correspondence should be addressed. MS 7550M. GOLA, C. DUCREUX AND H. CHAGNEUX successive Ca2+ entries. Thus, C-channels did not appear to be inactivated by depolarization or increase in [Ca2+]i.8. C-channels were found to be relatively highly voltage dependent, with an e-fold increase in PO per 14-9 mV increase in potential. The location of the PO-V curve on the voltage axis, but not its shape, was variable along a 60 mV span. Openings were never observed at potentials below -30 mV.9. Increasing Ca2+ influxes shifted the PO-V relationship towards negative potentials. Reverse shifts were observed during the PO decay following a brief Ca2+ entry.10. The latency to first opening was of 80-100 ms when patch depolarizations were applied immediately after a brief Ca2+ entry. The latency decreased to 10-20 ms when the depolarization was applied 300-600 ms after the Ca2+ entry. These data would indicate that the C-current activation was rate limited by calcium while the voltage-dependent step had fast kinetics.11. The distributions of open time, closed time and burst duration were con...

Large conductance Ca(2+)‐activated K+ channels are involved in both spike shaping and firing regulation in Helix neurones.

“…In spite of the slow time course of the macroscopic current, recordings of unitary currents in cell-attached patches have shown that the BK channel opening probability peaks within a few milliseconds of the spike upstroke at values as large as 0'25-04 (Lang & Ritchie, 1987;Gola et al 1990). These findings suggested that whole-cell BK currents observed under voltage clamp conditions did not give reliable information on their involvement in freely firing cells, presumably because of the way Ca2+ ions enter the cell in these different situations (see below).…”

“…Its opening is dependent on both voltage and calcium ). According to Kostyuk, Doroshenko & Tsyndrenko (1980) and Lux & Hofmeier (1982) (Muller, Swandulla & Lux, 1989;Gola et al 1990). In the following experiments we reevaluated these hypotheses by correlating the activation rate of the macroscopic KCa current with the size of the underlying Ca2+ current.…”

“…It results from the opening of large-conductance (> 100 pS) BK channels. Although the molluscan K+ channels described in Aplysia (Belardetti, Schacher & Siegelbaum, 1986) and Helix neurones (Gola, Ducreux & Chagneux, 1990) have a relatively small unitary conductance (20-65 pS), they actually share the main properties of the BK-type channels. The second K+a current is poorly voltage dependent, insensitive to TEA' and charybdotoxin and can be either apamin sensitive (rat and bullfrog sympathetic ganglia) or apamin insensitive (hippocampal neurones).…”

“…On the basis of whole-cell voltage and current recordings and of unitary channel properties it is generally assumed that C-or BK channels are involved in active potential repolarization (Adams, Constanti, Brown & Clark, 1982; Gola, Hussy, Crest & Ducreux, 1986;Storm, 1987;Lang & Ritchie, 1987;Gola et al 1990; Lancaster et al 1991) and play a limited role, if any, in post-spike events and in the spike frequency regulation. Since SK and related Ca2+-activated K+ channels have little voltage dependence and are more sensitive to calcium than BK channels, they are ideally suited for acting in post-spike events and therefore for contributing to the spike frequency regulation (Madison & Nicoll, 1984;Lancaster et al 1991).…”

Nitric oxide modulation of calcium‐activated potassium channels in postganglionic neurones of avian cultured ciliary ganglia

“…At the amphibian neuromuscular junction charybdotoxinsensitive channels normally decrease the duration of the terminal action potential and thereby determine the extent of calcium entry into the terminal, and therefore the amount of transmitter released (Robitaille & Charlton, 1992). This process is facilitated by the Ic channels being placed in the vicinity of the high density of calcium-channels that are strategically positioned at the release sites (Roberts et al, 1990;Gola et al, 1990;Robitaille & Charlton, 1992). NO blocks high-voltage threshold calcium channels in the ciliary ganglion (Khurana & Bennett, 1993) as well as blocking calcium influx in smooth muscle (Magliola & Jones, 1990;Clapp & Gurney, 1991).…”