Alphaxalone Activates a Cl− Conductance Independent of GABAA Receptors in Cultured Embryonic Human Dorsal Root Ganglion Neurons

Abstract

Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone’s (5α-pregnane-3α-ol-11,20-dione) effects on GABAAreceptors and on Cl− currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl− currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1–5.0 μM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 ± 0.15). The alphaxalone-induced whole cell current was carried largely by Cl− ions. Its reversal potential was close to the theoretical Cl− equilibrium potential, changing with a shift in the external Cl−concentration as predicted by the Nernst equation for Cl−ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 μM), it did not appear to result from activation of GABAAreceptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl− concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 μM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl− channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 μM alphaxalone revealed a single conductance state with a chord conductance of ∼29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl− channels whose electrophysiological and pharmacological properties are distinct from those of Cl−channels associated with GABAA receptors.