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Single channel currents at six microsecond resolution elicited by acetylcholine in mouse myoballs

Franz Parzefall, Robert Wilhelm, Manfred Heckmann and Josef Dudel

1. A patch-clamp set-up was optimized for low noise and high time resolution. An Axoclamp 200B amplifier was modified to incorporate a Teflon connector to the electrode. An electrode puller was equipped with a hydrogen-oxygen burner to produce quartz-glass pipettes with optimally 0·2 µm openings and 20 M resistance.

2. The r.m.s. (root mean square) noise of sealed pipettes in the bath ranged from 3·6 fA with 100 Hz filter cut-off to 1·5 pA with 61 kHz filter cut-off. At these extremes currents of 17 fA and more than 3 ms, or 9 pA and more than 6 µs could be resolved with a negligible error rate.

3. The system was tested on mouse myoballs, recording 9-10 pA single channel currents on-cell at -200 mV polarization which were elicited by 0·1-5000 µM acetylcholine (ACh).

4. Distributions of open and closed times and of correlations of open times to the preceding closed time defined several open states: single openings with mean durations of 1·2 and 25 µs, from single-liganded receptors, and bursts of 10 ms mean duration containing on average 800 µs openings and 16 µs closings, from double liganded receptors. Above 0·1 mM ACh these openings are interrupted increasingly by on average 18 µs and 72 µs channel blocks by ACh.

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