BACKGROUND AND PURPOSE The binding of transmitter to specialized binding pockets leads to rearrangements in the structure of the receptor eventually resulting in channel opening. We used voltage‐clamp fluorometry to investigate the pharmacological basis and biophysical processes that underlie structural changes at the transmitter binding site of the rat α1β2γ2L GABAA receptor.EXPERIMENTAL APPROACH Simultaneous electrophysiological and site‐specific fluorescence measurements were conducted on receptors expressed in Xenopus oocytes and labelled with an environmentally‐sensitive fluorophore, Alexa 546 maleimide, at the α1L127C site.KEY RESULTS Receptors activated by GABA demonstrate a concentration‐dependent increase in fluorescence intensity, indicating that the environment surrounding the fluorophore becomes less polar upon activation. Qualitatively similar responses were observed with other GABA site ligands such as piperidine‐4‐sulphonic acid, muscimol, β‐alanine and 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol. Fluorescence changes were not affected by the direction of current flow. During long applications of GABA significant desensitization developed, which was not accompanied by additional changes in fluorescence. Pentobarbital was an efficacious agonist of the labelled mutant receptor but did not cause changes in fluorescence. Direct activation by etomidate or the steroid allopregnanolone also did not result in fluorescence changes. Functional potentiation of GABA‐activated receptors by allopregnanolone or etomidate enhanced both the GABA‐elicited functional response and the fluorescence change. In contrast, potentiation by pentobarbital was not accompanied by an enhanced fluorescence response.CONCLUSIONS AND IMPLICATIONS The data indicate that there is no direct correlation between current flow or position of the activation gate and the structural changes as detected by Alexa 546‐labelled α1L127Cβ2γ2L GABAA receptors. Channel potentiation by pentobarbital qualitatively differs from potentiation by etomidate or allopregnanolone.

Ethanol (EtOH) tachyphylaxis (acute tolerance), a time‐dependent decrease in apparent potency, is known in vivo and in some neuronal preparations. The present studies characterize EtOH tachyphylaxis in spinal motorneurons and test the hypothesis that metabotropic glutamate receptors (mGluRs) play a role. Patch clamp studies were carried out in motorneurons in rat spinal cord slices. Currents were evoked by pulses of glutamate, alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) or N‐methyl‐D‐aspartic acid (NMDA). In nine of 15 cells, ethanol depression of glutamate‐evoked currents was time‐dependent. EtOH depressed current area 36.9±3% at 8–10 min, but only 16.8±3% at 20 min. Mean reduction in depression was 20.1±1%, N=9. Tachyphylaxis was less prominent in currents evoked by AMPA or NMDA, appearing in two of 10 AMPA and three of 11 NMDA currents. The mGluR agonist trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (ACPD) increased, the antagonist (±)‐alpha‐methyl‐4‐carboxyphenylglycine (MCPG) decreased the area of glutamate‐evoked currents. ACPD also increased the area of NMDA‐ and AMPA‐evoked currents. ACPD increased the incidence of tachyphylaxis in glutamate‐evoked currents to 100% (N=9); MCPG markedly reduced tachyphylaxis. ACPD also increased the incidence of tachyphylaxis in currents evoked by NMDA and AMPA to five of eight and four of seven neurons, respectively. Block of G‐protein pathways by intracellular GDP‐β‐s abolished tachyphylaxis in glutamate‐evoked currents (N=8); however, currents recovered only partially following EtOH washout. Activation of mGluRs contributes to neuronal tachyphylaxis to EtOH in spinal cord motorneurons, probably via G‐protein pathways. British Journal of Pharmacology (2003) 138, 1417–1424. doi:10.1038/sj.bjp.0705175

Ethanol is a general anesthetic agent as defined by abolition of movement in response to noxious stimulation. This anesthetic endpoint is due to spinal anesthetic actions. This study was designed to test the hypothesis that ethanol acts directly on motor neurons to inhibit excitatory synaptic transmission at glutamate receptors. Whole cell recordings were made in visually identified motor neurons in spinal cord slices from 14- to 23-day-old rats. Currents were evoked by stimulating a dorsal root fragment or by brief pulses of glutamate. Ethanol at general anesthetic concentrations (50-200 mM) depressed both responses. Ethanol also depressed glutamate-evoked responses in the presence of tetrodotoxin (300 nM), showing that its actions are postsynaptic. Block of inhibitory gamma-aminobutyric acidA and glycine receptors by bicuculline (50 microM) and strychnine (5 microM), respectively, did not significantly reduce the effects of ethanol on glutamate currents. Ethanol also depressed glutamate-evoked currents when the inhibitory receptors were blocked and either D, L-2-amino-5-phosphonopentanoic acid (40 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione disodium (10 microM) were applied to block N-methyl-D-aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. The results show that ethanol exerts direct depressant effects on both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate glutamate currents in motor neurons. Enhancement of gamma-aminobutyric acidA and glycine inhibition is not required for this effect. Direct depression of glutamatergic excitatory transmission by a postsynaptic action on motor neurons thus may contribute to general anesthesia as defined by immobility in response to a noxious stimulus.