Multiple mechanisms of block by the anesthetic isoflurane of a γ-aminobutyric acid activated chloride channel in crayfish

Abstract

Outside-out patches were excised from the membrane of the deep extensor abdominal muscle (DEAM), containing γ-aminobutyric acid (GABA)- activated chloride channels, in the crayfish Astacus astacus. GABA and isoflurane (iso) were applied in pulses by a liquid filament switch, and their effects on the GABA-elicited chloride currents were investigated. Application of iso alone elicited no current responses and pre-application of iso prior to GABA had no effects on the GABA-elicited current. Co-application of GABA and iso resulted in a reduction of the initial chloride current and subsequent decline of the current to a steady state, indicating that iso binds to the receptor after GABA has bound. Recovery currents at the end of the co-application pulse, their amplitudes decreasing with pulse duration, confirmed this suggestion. Open-time distributions of the blocked channel showed a shift of the long open-time towards a new time constant, indicating a second block mechanism via the long open state A₅O_s of the channel. Removal of GABA and iso after reaching the equilibrium state of the block resulted in recovery currents containing exclusively openings from the long open state A₅O_s, confirming the suggestion of an open channel block only at one of the open states.