Alphaxalone activates a Cl- conductance independent of GABAA receptors in cultured embryonic human dorsal root ganglion neurons.
Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha-pregnane-3alpha-ol-11,20-dione) effects on GABAA receptors 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 microM) 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 microM), it did not appear to result from activation of GABAA receptors. 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 microM 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 microM alphaxalone revealed a single conductance state with a chord conductance of approximately 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.
Neurophysiology and Spinal Cord Pharmacology Laboratories, Veterans Affairs Medical Center, Miami, Florida 33101, USA.
A Y
AY
Valeyev
J C
JC
Hackman
A M
AM
Holohean
P M
PM
Wood
J L
JL
Katz
R A
RA
Davidoff
D000777:Anesthetics / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D004622:Embryo, Mammalian; D005727:Ganglia, Spinal / Q000166:cytology / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011277:Pregnanediones / Q000494:pharmacology*; D018079:Receptors, GABA / Q000187:drug effects / Q000502:physiology; D011963:Receptors, GABA-A / Q000187:drug effects / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000777:Anesthetics; D019086:Bridged Bicyclo Compounds, Heterocyclic; D018118:Chloride Channels; C087200:GABA-C receptor; D011277:Pregnanediones; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D010566:Virulence Factors, Bordetella; C037476:tert-butylbicyclophosphorothionate; C006477:alphaxalone