Single channel currents at six microsecond resolution elicited by acetylcholine in mouse myoballs.
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 micron openings and 20 MOmega 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 micros 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 microM 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 micros, from single-liganded receptors, and bursts of 10 ms mean duration containing on average 800 micros openings and 16 micros closings, from double liganded receptors. Above 0.1 mM ACh these openings are interrupted increasingly by on average 18 micros and 72 micros channel blocks by ACh.
Parzefall
F
F
Institut fur Physiologie der Technischen Universitat Munchen, Biedersteinerstrasse 29, D-80802 Munich, Germany.
Wilhelm
R
R
Heckmann
M
M
Dudel
J
J
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D004558:Electric Stimulation / Q000295:instrumentation / Q000379:methods; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D011950:Receptors, Cholinergic / Q000502:physiology*; D012680:Sensitivity and Specificity; D013997:Time Factors
D007473:Ion Channels; D011950:Receptors, Cholinergic; D000109:Acetylcholine