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

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 A5Os 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 A5Os, confirming the suggestion of an open channel block only at one of the open states.