Activation and desensitization of embryonic-like receptor channels in mouse muscle by acetylcholine concentration steps

Abstract

1. Pulses of acetylcholine (ACh) in concentrations between 0.1 and 1000 microM were applied repetitively to outside-out patches of enzymatically denervated (14 days) mouse muscle with the liquid filament switch. Solutions superfusing the patch could be changed rapidly (within 0.2 ms). 2. Single-channel activity was studied under steady-state conditions in the outside-out and in the cell-attached mode. The single-channel conductance was 26 pS in outside-out patches, characteristic for embryonic-like channels. Apparent mean open time was about 2.5 ms, a shorter component of closed times was 800 microseconds and burst length was about 5 ms. 3. Channel currents elicited by pulses of ACh were averaged. The time-to-peak current was concentration dependent and decreased from a level of about 10 ms below 10 microM to about 400 microseconds at 100 microM-ACh. 4. For a typical experiment, the average peak current, imax, increased from -0.4 pA with 0.1 microM to -82 pA with 1000 microM-ACh, close to the value at saturation. The half-maximal response was at 60 microM-ACh. The dose-response curves for imax had double-logarithmic slopes of 1.1-1.3, consistent with two binding sites at the embryonic nicotinic acetylcholine receptor (nAChR). 5. The current elicited by ACh pulses decreased rapidly after the peak. The time constant of desensitization increased from 20-50 ms with 1000 microM-ACh to up to more than a second with 1 microM-ACh. 6. The current in steady state (fully desensitized) increased up to 10 microM-ACh, but decreased slightly to values of imax/100 to imax/500 when higher concentrations were applied. 7. In addition to the well-known differences between adult and embryonic nAChR concerning the apparent mean open time and burst length, we found differences in the slope of the dose-response curve for imax, in the ratio of peak to steady-state response, and in the rise time of the response.