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...