Single channel currents from excised patches of muscle membrane.

Abstract

The currents through single acetylcholine-activated channels were measured on membrane fragments that were torn from rat muscle myotubes with patch pipettes. The membrane fragments were sealed into the pipette by using the gigohm-seal technique which permitted voltage clamp of the membrane via the patch electrode. Membrane patches were excised by sudden withdrawal of the electrode from the cell. Substitution of fluoride for chloride ions in the bathing solution could prevent or reverse the tendency for the membrane at the electrode tip to seal over into a closed vesicle. The single membrane layer at the electrode tip could remain functional for up to 30 min. The apparent single channel conductance was minimally affected by excision. The current-voltage relationships for the single channel currents show that the inside (i.e., cytoplasmic surface) of the membrane fragment was exposed to the bathing solution. In symmetric Na solutions the current-voltage curve was nearly linear and reversed at approximately 0 mV. In other bathing solutions from 40-500 mM NaF, the observed zero current potential was close to that predicted by the Nernst equation. Evidence is presented that internal Na interacts with the channel, causing both saturation of outward current and block of inward current. At +100 mV the apparent dissociation constant for internal Na was 138 mM.