SUMMARY1. Single channel currents through acetylcholine receptor channels (ACh channels) were recorded at chronically denervated frog muscle extrajunctional membranes in the absence and presence of the lidocaine derivatives QX-222 and QX-314.2. The current wave forms due to the opening and closing of single ACh channels (activated by suberyldicholine) normally are square pulses. These single pulses appear to be chopped into bursts of much shorter pulses, when the drug QX-222 is present in addition to the agonist.3. The mean duration of the bursts is comparable to or longer than the normal channel open time, and increases with increasing drug concentration.4. The duration of the short pulses within a burst decreases with increasing drug concentration.5. It is concluded that drug molecules reversibly block open end-plate channels and that the flickering within a burst represents this fast, repeatedly occurring reaction.6. The voltage dependence of the reaction rates involved, suggested that the site of the blocking reaction is in the centre of the membrane, probably inside the ionic channel.

No abstract

GABA A receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABA A receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABA A receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at mu-molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10--100ms. In order to obtain the necessary resolution, a delicate compromise had to be found between different experimental parameters. Pipettes with 1--3 micrometer internal diameter and a steep final taper had to be used, extensive enzyme treatment was necessary, and conditions had be to found in which channels open at a relatively low frequency.

Anesthetic drugs are known to interact with GABA A receptors, both to potentiate the effects of low concentrations of GABA and to directly gate open the ion channel in the absence of GABA; however, the site(s) involved in direct gating by these drugs is not known. We have studied the ability of alphaxalone (an anesthetic steroid) and pentobarbital (an anesthetic barbiturate) to directly activate recombinant GABA A receptors containing the ␣1, ␤2, and ␥2L subunits. Steroid gating was not affected when either of two mutated ␤2 subunits [␤2(Y157S) and ␤2(Y205S)] are incorporated into the receptors, although these subunits greatly reduce the affinity of GABA binding. These observations indicate that steroid binding and subsequent channel gating do not require these particular residues, as already shown for barbiturates. Bicuculline or gabazine (two competitive antagonists of GABA binding) reduced the currents elicited by alphaxalone and pentobarbital from wild-type GABA A receptors; however, gabazine produced only a partial block of responses to pentobarbital or alphaxalone, and bicuculline only partially blocked responses to pentobarbital. These observations indicate that the blockers do not compete with alphaxalone or pentobarbital for a single class of sites on the GABA A receptor. Finally, at receptors containing ␣1␤2(Y157S)␥2L subunits, both bicuculline and gabazine showed weak agonist activity and actually potentiated responses to alphaxalone. These observations indicate that the blocking drugs can produce allosteric changes in GABA A receptors, at least those containing this mutated ␤2 subunit. We conclude that the sites for binding steroids and barbiturates do not overlap with the GABA-binding site. Furthermore, neither gabazine nor bicuculline competes for binding at the steroid or barbiturate sites. The data are consistent with a model in which both gabazine and bicuculline act as allosteric inhibitors of channel opening for the GABA A receptor after binding to the GABA-binding site.Key words: GABA A receptor; GABA; neurosteroids; bicuculline; inverse agonist; anesthetics; allosteric inhibitor GABA activates a ligand-gated ion channel (the GABA A receptor), which underlies most rapid inhibition in the brain. Various other compounds also bind to the GABA A receptor and can gate the channel or modulate channel function (Macdonald and Olsen, 1993). In particular, steroids and barbiturates are each able to directly gate the GABA A receptor channel (in the absence of GABA), and they can also enhance the activation produced by low concentrations of GABA. It is not known whether the same sites are involved in producing direct gating and in potentiating the effects of GABA. For the sites involved in potentiation, however, the steroid-binding site and the barbiturate-binding site are distinct from each other and are also distinct from the GABAbinding site (Macdonald and Olsen, 1993). Because the characterized sites for steroid and barbiturate binding differ from the GABA-binding site, it is puzzling that a competit...

We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR.

Endogenous neurosteroids have rapid actions on ion channels, particularly GABA(A) receptors, which are potentiated by nanomolar concentrations of 3alpha-hydroxypregnane neurosteroids. Previous evidence suggests that 3beta-hydroxypregnane steroids may competitively antagonize potentiation induced by their 3alpha diastereomers. Because of the potential importance of antagonists as experimental and clinical tools, we characterized the functional effect of 3beta-hydroxysteroids. Although 3beta-hydroxysteroids reduced the potentiation induced by 3alpha-hydroxysteroids, 3beta-hydroxysteroids acted noncompetitively with respect to potentiating steroids and inhibited the largest degrees of potentiation most effectively. Potentiation by high concentrations of barbiturates was also reduced by 3beta-hydroxysteroids. 3beta-Hydroxysteroids are also direct, noncompetitive GABA(A) receptor antagonists. 3beta-Hydroxysteroids coapplied with GABA significantly inhibited responses to > or =15 microm GABA. The profile of block was similar to that exhibited by sulfated steroids, known blockers of GABA(A) receptors. This direct, noncompetitive effect of 3beta-hydroxysteroids was sufficient to account for the apparent antagonism of potentiating steroids. Mutated receptors exhibiting decreased sensitivity to sulfated steroid block were insensitive to both the direct effects of 3beta-hydroxysteroids on GABA(A) responses and the reduction of potentiating steroid effects. At concentrations that had little effect on GABAergic synaptic currents, 3beta-hydroxysteroids and low concentrations of sulfated steroids significantly reversed the potentiation of synaptic currents induced by 3alpha-hydroxysteroids. We conclude that 3beta-hydroxypregnane steroids are not direct antagonists of potentiating steroids but rather are noncompetitive, likely state-dependent, blockers of GABA(A) receptors. Nevertheless, these steroids may be useful functional blockers of potentiating steroids when used at concentrations that do not affect baseline neurotransmission.

Neuroactive steroids have some of their most potent actions by augmenting the function of GABA A receptors. Endogenous steroid actions on GABA A receptors may underlie important effects on mood and behavior. Exogenous neuroactive steroids have potential as anesthetics, anticonvulsants, and neuroprotectants. We have taken multiple approaches to understand more completely the interaction of neuroactive steroids with GABA A receptors. We have developed many novel steroid analogues in this effort. Recent work has resulted in synthesis of new enantiomer analogue pairs, novel ligands that probe various properties of the steroid pharmacophore, fluorescent neuroactive steroid analogues, and photoaffinity labels. Using these tools, combined with receptor binding and electrophysiological assays, we have begun to untangle the complexity of steroid actions at this important class of ligand-gated ion channel.