Randomized dose-controlled study of topiramate as first-line therapy in epilepsy.

OBJECTIVES: To evaluate the efficacy and tolerability of topiramate as monotherapy, using a dose-controlled study design. MATERIALS AND METHODS: We conducted a multinational, randomized, double-blind trial in adults and children (> or =6 years old) with epilepsy that was not being treated when randomized to 400 or 50 mg/day topiramate as target maintenance dosages. In addition to > or =2 lifetime unprovoked seizures, patients had to have one or two partial-onset seizures or generalized-onset tonic-clonic seizures in the 3-month retrospective baseline. The primary efficacy end point was time to first seizure; a secondary efficacy measure was the seizure-free rate at 6 months and 1 year. Double-blind treatment continued until 6 months after the last patient was randomized. RESULTS: Kaplan-Meier survival analyses for time to first seizure (intent-to-treat, n = 470) favored 400 mg/day over 50 mg/day (P = 0.0002) as a target maintenance dosage. The first evaluation point with a significant difference (P = 0.046) favoring the higher dose was at day 14 when patients were receiving 100 or 25 mg/day. The probability of being seizure-free at 6 months was 83% in patients randomized to 400 mg/day and 71% in those randomized to 50 mg/day (P = 0.005). Seizure-free rates at 12 months were 76% and 59%, respectively (P = 0.001). Differences favoring the higher dose were significant in patients with partial-onset seizures (P = 0.009) and in those with generalized-onset tonic-clonic seizures (P = 0.005). The most common dose-related adverse events were paresthesia, weight loss, and decreased appetite. Discontinuations due to cognitive-related adverse events were 2% in the 50-mg group and 7% in the 400-mg group. Overall, 7% and 19%, respectively, discontinued with adverse events during the median treatment duration of 9 months. CONCLUSION: Topiramate is effective as monotherapy in adults and children. Because a therapeutic effect emerges during titration, clinicians should adjust dosages in step-wise fashion with intermediate stopping points, e.g., 100 mg/day, to evaluate patient response and achieve the optimal maintenance dosage.

The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor.

Ionotropic GABA receptors generally require the products of three subunit genes. By contrast, the GABA receptor needed for locomotion in Caenorhabditis elegans requires only the unc-49 gene. We cloned unc-49 and demonstrated that it possesses an unusual overlapping gene structure. unc-49 contains a single copy of a GABA receptor N terminus, followed by three tandem copies of a GABA receptor C terminus. Using a single promoter, unc-49 generates three distinct GABAA receptor-like subunits by splicing the N terminus to each of the three C-terminal repeats. This organization suggests that the three UNC-49 subunits (UNC-49A, UNC-49B, and UNC-49C) are coordinately rescued and therefore might coassemble to form a heteromultimeric GABA receptor. Surprisingly, only UNC-49B and UNC-49C are expressed at high levels, whereas UNC-49A expression is barely detectable. Green fluorescent protein-tagged UNC-49B and UNC-49C subunits are coexpressed in muscle cells and are colocalized to synaptic regions. UNC-49B and UNC-49C also coassemble efficiently in Xenopus oocytes and HEK-293 cells to form a heteromeric GABA receptor. Together these data argue that UNC-49B and UNC-49C coassemble at the C. elegans neuromuscular junction. Thus, C. elegans is able to encode a heteromeric GABA receptor with a single locus.

Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors.

1. Regional distinctions in GABA type A (GABAA) miniature IPSC responses are thought to be determined by postsynaptic receptor composition. The kinetics of receptor activation and deactivation were studied using rapid exchange (100 micros) of GABA at excised patches containing recombinant (alpha1beta1gamma2 or alpha2beta1gamma2) and native (cortical) GABAA receptors. 2. Receptors activated by brief (< 1 ms) pulses of GABA demonstrated a characteristic current response, hereby referred to as the 'receptor system response'. System response properties included agonist concentration-dependent peak amplitudes and concentration-independent maximal rates of activation and deactivation. Receptor subtypes were characterized functionally and phenotyped using the system response characteristics. 3. System responses obtained for alpha1beta1gamma2 receptors exhibited a single phenotype while alpha2beta1gamma2 receptors exhibited either a predominant slow deactivation (type I) or a relatively infrequent faster (type II) phenotype. Receptor system responses of alpha2beta1gamma2 receptors reached peak currents twice as fast as those of alpha1beta1gamma2 receptors (0.5 versus 1.0 ms) but decayed 2 or 6 times more slowly (taulong of approximately 190 and 62 ms for type I and II alpha2beta1gamma2, and approximately 34 ms for alpha1beta1gamma2 receptors). 4. Receptor system responses from cultured fetal mouse cortical neurons could be statistically separated and classified into five major types with little intragroup variability, primarily based on variations in the current deactivation phases. 5. Receptors subjected to pharmacological modulation exhibited alterations in system response properties consistent with known mechanisms of action, such that distinctions between binding and gating modulations were possible. 6. Brief agonist exposure places limits on receptor activation and deactivation response kinetics. Consequently, receptor system responses may be used to characterize and functionally phenotype an excised patch receptor population. Furthermore, since synaptic exposure to transmitter is postulated to be similarly brief, IPSC kinetics may reflect a functional fingerprint of synaptic receptors.

The methylglutamate, SYM 2081, is a potent and highly selective agonist at kainate receptors.

The methylglutamate analog (2S,4R)-4-methylglutamate (SYM 2081) has been shown to potently displace high affinity [3H]kainate binding to cortical tissue and to recombinant kainate receptors, and to evoke rapidly desensitizing responses in electrophysiological recordings. We have used two electrode voltage clamp recordings to compare the potency and efficacy of SYM 2081 with other alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor agonists at homomeric kainate and AMPA receptors expressed in Xenopus oocytes. In the presence of concanavalin A to reduce agonist induced desensitization at kainate receptors, SYM 2081 was a potent agonist at homomeric kainate receptors composed of the GluR5 and GluR6 subunit, with an EC50 of 0.12 +/- 0.02 and 0.23 +/- 0.01 microM, respectively. SYM 2081 was highly selective for kainate receptors, the EC50 for activation of AMPA receptors composed of the GluR1 and GluR3 subunits was 132 +/- 44 and 453 +/- 57 microM, respectively. Other methylglutamate analogs were tested for kainate receptor agonist activity. Methylglutamate compounds with the methyl group at the 2 or 3 position of glutamate were inactive indicating that positioning of the methyl group at the 4 position was essential for agonist activity. Of the four stereoisomers of 4-methylglutamate, SYM 2081 (2S,4R) was the most potent agonist. The (2R,4R) isomer was estimated to be 20-fold and the (2S,4S)-isomer approximately 1000-fold less potent than SYM 2081. These results indicate that SYM 2081 is a potent and selective agonist at kainate receptors, and thus will be a useful ligand for evaluating the role of kainate receptors in central nervous system function and disease.

Activation and deactivation rates of recombinant GABA(A) receptor channels are dependent on alpha-subunit isoform.

The role of subunit composition in determining intrinsic maximum activation and deactivation kinetics of GABA(A) receptor channels is unknown. We used rapid ligand application (100-micros solution exchange) to examine the effects of alpha-subunit composition on GABA-evoked activation and deactivation rates. HEK 293 cells were transfected with human cDNAs encoding alpha1beta1gamma2- or alpha2beta1gamma2-subunits. Channel kinetics were similar across different transfections of the same subunits and reproducible across several GABA applications in the same patch. Current rise to peak was at least twice as fast for alpha2beta1gamma2 receptors than for alpha1beta1gamma2 receptors (reflected in 10-90% rise times of 0.5 versus 1.0 ms, respectively), and deactivation was six to seven times slower (long time constants of 208 ms versus 31 ms) after saturating GABA applications. Thus alpha-subunit composition determined activation and deactivation kinetics of GABA(A) receptor channels and is therefore likely to influence the kinetics and efficacy of inhibitory postsynaptic currents.

Identification of amino acids in the glutamate receptor, GluR3, important for antibody-binding and receptor-specific activation.

We reported (Twyman, R. E., Gahring, L. C., Speiss, J., and Rogers, S. W. (1995) Neuron 14, 755-762) that antibodies to a subregion of the glutamate receptor (GluR) subunit GluR3 termed GluR3B (amino acids 372-395), act as highly specific GluR agonists. In this study we produced additional rabbit anti-GluR3B-specific antibodies, ranked them according to their ability to function as GluR agonists and characterized the immunoreactivity using deletion and alanine substitution mutagenesis. These anti-GluR3B antibodies bound to a subset of the residues in GluR3B (amino acids 372-386), of which glutamate 375, valine 378, proline 379, and phenylalanine (Phe) 380 were preferred. The level of GluR activation correlated with the binding of antibody to Phe-380, which suggests that immunoreactivity directed toward Phe-380 is an index for the anti-GluR agonist potential. Since the identity of this residue varies between respective GluR subunits, this suggested that this residue may be important for imparting antibody subunit specificity. To test this possibility, the alanine in GluR1 was converted to a phenylalanine, which extended the subunit specificity from GluR3 to the modified GluR1. We conclude that antibody contacts with key residues in the GluR3B region define a novel GluR subunit-specific agonist binding site and impart subunit-specific immunoreactivity.

Autoantibodies to glutamate receptor subunit GluR2 in nonfamilial olivopontocerebellar degeneration.

We describe a 63-year-old man with a 5-year history of progressive sporadic olivopontocerebellar atrophy (OPCA) who exhibits high serum titers of IgM autoantibodies to the neuronal glutamate receptor subunit GluR2. Immunohistochemistry revealed intense staining of mouse cerebellar Purkinje cells and cells in the pontine nuclei and olivary complex. Glutamate receptor currents were activated in a subset of cultured mouse neurons by an anti-GluR2 IgM fraction, and they were blocked by the competitive AMPA-type glutamate receptor antagonist CNQX and by a synthetic peptide to a specific epitope region of GluR2 (AA 369-393). The patient was treated with nine courses of plasmapheresis with little improvement of symptomatology. However, IgM titers to GluR2 decreased approximately 8-fold and the serum functional activity decreased proportionally. These findings may suggest a role for autoimmunity to glutamate receptors in the pathophysiology of certain forms of progressive nervous system degeneration.

The role of autoimmunity to glutamate receptors in neurological disease.

Understanding and experimentally approaching the processes that underlie the origin and progression of many severe neurological disorders presents a challenge to both clinical and basic researchers. We have found that the origin of some neurological diseases, including a rare form of childhood epilepsy and a neurodegenerative disease associated with peripheral cancers, may be related to production of physiologically active autoantibodies that are directed towards excitatory ionotropic glutamate receptors of the brain. This suggests that some neurological diseases may result from dysfunction of the immune system.

Direct evidence for diazepam modulation of GABAA receptor microscopic affinity.

Alteration of agonist affinity is a potential mechanism for pharmacological modulation of ligand-gated receptor channel function. The time course for receptor activation and current onset is determined by the combined rates for two kinetic transitions that underlie the protein confirmations for binding agonist and channel gating. Using ultrafast ligand exchange techniques, we distinguish between these previously difficult to separate events and demonstrate their independent pharmacological modulation. Diazepam, which increases apparent affinity of gamma-aminobutyric acid (GABA) to GABAA receptors, was used to examine its effects on GABA binding and ion channel gating of expressed alpha 2 beta 1 gamma 2 receptors from excised outside-out patches of acutely transfected HEK 293 cells. Diazepam increased rates of current onset evoked by low concentrations (< 1 mM) but not at saturating GABA concentrations. Furthermore, rates of current decay were not affected during brief applications of GABA, and thus, demonstrated a diazepam specific effect on ligand binding affinity and not channel gating kinetics. However, current decay during and following prolonged GABA applications were altered by diazepam in a fashion similar to that for higher concentrations of GABA which also increased receptor desensitization. These findings and analysis by computer modeling indicated that diazepam likely enhances GABA receptor currents primarily by accelerating GABA association to its receptor at the first agonist binding site. These results provide the first direct physiological evidence for pharmacological modulation of microscopic binding affinity of GABA receptors.

Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site.

Two rabbits immunized with a portion of glutamate receptor (GluR) subunit GluR3 (amino acids 245-457) exhibited seizure-like behaviors, suggesting that antibodies to GluR3 may modulate neuronal excitability. Using whole-cell recording, rabbit GluR3 antisera were tested on cultured fetal mouse cortical neurons. In a subset of kainate-responsive neurons, miniperfusion of antisera and IgG evoked currents that were blocked by CNQX. Immunoreactivity to synthetic peptides prepared to subregions GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395) was present in both rabbit sera. Peptide GluR3B, but not GluR3A, specifically blocked antisera- and IgG-evoked currents. Similar receptor activation and anti-GluR3 reactivity was present in sera from patients with active Rasmussen's encephalitis, an intractable pediatric epilepsy. Thus, antibodies to GluR3 define a region involved in agonist binding and specific receptor activation. These data suggest that antibodies to neuronal receptors can function as agonists and that autoantibodies to GluRs may be highly specific neurotoxicants in some neurological diseases.

Autoantibodies to neuronal glutamate receptors in patients with paraneoplastic neurodegenerative syndrome enhance receptor activation.

BACKGROUND: Paraneoplastic syndromes are "remote" complications of cancer characterized clinically by neurological disease. The sera and cerebrospinal fluid (CSF) from patients with paraneoplastic neurological syndromes (PNS) frequently contain autoantibodies to ill-defined neuronal antigens. We report here that neuronal glutamate receptors are targets for autoantibodies found in the serum from some patients with well-characterized PNS. MATERIALS AND METHODS: We have analyzed the serum from seven patients with well-characterized PNS for the presence of autoreactive antibodies to non-NMDA glutamate receptor subunits. Autoantibodies were assessed using Western blot, immunohistochemistry, and immunocytochemistry. Whole-cell electrophysiological recordings were used to examine the effect of antibodies on glutamate receptors expressed by cortical neurons in culture. RESULTS: Six of seven patients' serum contained autoantibodies to the non-NMDA glutamate receptor (GluR) subunits GluR1, GluR4, and/or GluR5/6. No patient had autoantibodies to GluR2, and only one patient exhibited weak immunoreactivity to GluR3. Electrophysiological analysis demonstrated that the serum from four of the six GluR-antibody-positive patients enhanced glutamate-elicited currents on cultured cortical neurons but had no effect on receptor function alone. Enhancement of glutamate-elicited currents was also produced by affinity-purified antibody to GluR5. CONCLUSIONS: The occurrence of autoantibodies to specific neuronal neurotransmitter subunits in the sera of patients with PNS and the ability of these autoantibodies to modulate glutaminergic receptor function suggest that some paraneoplastic neurological injury could result from glutamate-mediated excitotoxicity.

The dihydropyridine nitrendipine reduces N-methyl-D-aspartate (NMDA)-evoked currents of rodent cortical neurons through a direct interaction with the NMDA receptor-associated ion channel.

The 1,4-dihydropyridine (DHP) nitrendipine was previously shown to concentration-dependently (0.1-1 microM) reduce N-methyl-D-aspartate (NMDA)-evoked calcium influx and single-channel activity of mouse cerebellar granule cells and to reduce [3H]dizocilpine (MK-801) binding to mouse cortical and hippocampal membranes. Using patch-clamp electrophysiology, the present study was designed to understand further the specific mechanism of interaction between nitrendipine and NMDA receptors. Experiments were conducted with primary cultures of rodent cortical neurons and utilized whole-cell and excised outside-out patch configurations. NMDA-evoked whole-cell currents were reduced by nitrendipine (1 microM) in a voltage- and an agonist-dependent manner suggesting that nitrendipine interacts with NMDA receptors by a mechanism similar to that described for open channel blockers, such as extracellular magnesium and the dissociative anesthetics (e.g., MK-801). To examine this further, the effects of nitrendipine on NMDA-evoked single-channel activity were quantitated from outside-out patch recordings. In these studies, nitrendipine reduced the frequency of openings and bursts, reduced the average duration of openings and bursts and reduced the single open time constant for the main conductance (48 pS) in a concentration (0.03-1 microM)- and voltage-dependent manner. Because these effects of nitrendipine on NMDA-evoked currents were not readily reversible, the rate of nitrendipine dissociation is probably slower than the rate of NMDA-activated channel closing. Nitrendipine did not alter the main channel conductance at any concentration tested. Based on these results, a kinetic model of interaction between nitrendipine and NMDA receptors is proposed that is most similar to that previously described for MK-801.

Benzodiazepine and beta-carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.

1. The effects of the benzodiazepine receptor agonist, diazepam (DZ), and the inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), on gamma-aminobutyric acid (GABAA) receptor single channel currents were characterized. Outside-out patches were obtained from somata of cultured mouse spinal cord neurones and voltage clamped at -75 mV (ECl = 0 mV). 2. GABA (2 microM) alone or with DZ (20-1000 nM) or DMCM (20-100 nM) was applied to patches by pressure ejection from blunt micropipettes. DZ enhanced GABAA receptor currents with an inverted U-shaped concentration-response curve. Mean steady-state currents were increased by low concentrations of DZ (20-50 nM). At higher concentrations of DZ, the enhancement was diminished. Mean steady-state currents were decreased by DMCM at all concentrations. 3. GABAA receptor channels opened most frequently to a 27 pS main conductance level and less frequently to a 19 pS subconductance level. Neither DZ nor DMCM altered the proportion of time spent at either of the conductance levels. The kinetic properties of the main conductance level were studied. 4. Neither DZ nor DMCM altered the mean GABAA receptor channel open or burst durations. Sums of three exponential functions were required to fit best open and burst duration-frequency histograms for GABA alone or with DZ or DMCM. No significant changes in the three time constants or areas of the three exponential functions for open or burst duration histograms were produced by DZ or DMCM. 5. With increasing concentrations of DZ up to 50 nM, GABA evoked an increased frequency of channel openings and bursts. With higher DZ concentrations, the magnitudes of the increase in channel opening and burst frequencies were reduced. At all concentrations of DMCM, GABA evoked a decreased frequency of channel openings and bursts. 6. Closed duration-frequency histograms for GABA alone or with DZ or DMCM were best fitted by sums of at least six exponential functions. The three shortest closed duration time constants were unchanged by DZ or DMCM. The three longest closed duration time constants were altered by DZ and DMCM, consistent with alterations in opening frequency. 7. DZ increased and DMCM decreased steady-state GABAA receptor current by increasing or decreasing channel opening frequency without altering mean channel open duration. We propose that DZ and DMCM alter GABAA receptor current by acting reciprocally to increase or decrease only, respectively, the apparent agonist association rate at the first of two proposed GABA binding steps without altering channel gating.(ABSTRACT TRUNCATED AT 400 WORDS)

GABA-A receptor channel opening rates in excised patches of rat [correction of mouse] cortical neurons.

Patch clamp electrophysiology techniques and an ultrafast ligand exchange system (100 microseconds exchange time) were used to study opening rates of GABA-A receptor chloride channels in excised outside-out patches of fetal rat cortical neurons in culture. Receptor activation kinetics were studied using GABA (0.5 microM-10 mM) using rapid ligand exchanges. Patches contained 5-25 channels and were voltage clamped at -75 mV in symmetrical chloride solutions at room temperature. Responses were analyzed individually and as ensemble averages for each patch. Current rise times (10-90%) decreased with increased GABA concentration to a minimum of about 0.5 ms at 1 and 10 mM. Rates of channel activation at saturating GABA concentrations were similar to that recorded for miniature inhibitory currents at GABA synapses in these neurons. These data suggest that the synaptic concentration of GABA to activate GABA-A receptors in these neurons is at least 1 mM.

The dihydropyridine nitrendipine modulates N-methyl-D-aspartate receptor channel function in mammalian neurons.

Nitrendipine and other dihydropyridine voltage-sensitive calcium channel (VSCC) antagonists have been demonstrated to possess anticonvulsant and neuroprotectant activity in a variety of model systems. Likewise, antagonists of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype act as potent anticonvulsant and neuroprotective agents. Both VSCC and NMDA antagonists exert their effects by inhibiting the neuronal influx of calcium associated with activation of VSCCs or the NMDA receptor, respectively. Although results that provide evidence for crossreactivity between compounds acting at dihydropyridine-sensitive VSCCs and the NMDA receptor-channel complex have been reported, direct modulation of NMDA receptor function by dihydropyridines has not been demonstrated. In the present investigation, we report that nanomolar concentrations of nitrendipine reduced NMDA/glycine-evoked calcium flux and single-channel current in mouse cerebellar granule cell cultures. As measured with the calcium-specific probe indo-1, nitrendipine (0.1-10 microM) attenuated inward calcium flux evoked by bath application of NMDA (100 microM) and glycine (100 microM), in a concentration-dependent (IC50, 0.56 +/- 0.21 microM; 95% confidence interval, 0.19-1.3 microM) and reversible manner. Over a similar concentration range (0.01-100 microM), nitrendipine also inhibited the binding of [3H]MK-801 to mouse cortical and hippocampal membranes (IC50, 0.56 +/- 0.12 microM; 95% confidence interval, 0.37-0.84 microM). Finally, nitrendipine concentration- and voltage-dependently reduced the frequency of NMDA (10 microM)- and glycine (1 microM)-evoked single-channel openings and bursts recorded from excised outside-out patches of mouse cerebellar granule cells. These results indicate that nitrendipine suppresses NMDA/glycine-mediated calcium influx by a rapid and direct interaction with the NMDA receptor-channel complex. Furthermore, these results suggest that the interaction of nitrendipine with the NMDA receptor-channel complex is not tissue specific and probably does not require participation of calcium-dependent second messenger systems. Together, the data presented here support the novel hypothesis that nitrendipine may exhibit anticonvulsant and neuroprotectant activity via the combined ability to modulate both NMDA-associated ion channels and L-type VSCCs.

Kinetic properties of alpha 1 beta 1 gamma-aminobutyric acidA receptor channels expressed in Chinese hamster ovary cells: regulation by pentobarbital and picrotoxin.

Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ovary cells stably transfected with plasmids containing bovine gamma-aminobutyric acid (GABA) type A (GABAA) receptor channel alpha 1 and beta 1 subunit cDNAs. The predominant or main conductance level recorded had a 17-pS chord conductance. There were minor contributions made from 25-pS and 11-pS conductance levels. Average open duration, burst duration, and openings/burst did not change as the GABA concentration was increased from 5 to 25 microM. However, opening frequency increased from 11.0 to 19.5 openings/sec. Pentobarbital increased average channel open duration without increasing opening frequency, whereas picrotoxin slightly reduced average channel open duration and reduced opening frequency. Open duration frequency distributions were fitted best with the sum of two exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had at least two open states. The time constants and relative proportions of the two components did not vary when GABA concentration was increased from 5 to 25 microM. Closed duration distributions of closures between main conductance level openings were fitted best with multiple exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had several closed states. Burst duration frequency distributions were fitted best with two exponential functions whose time constants and relative proportions did not change with GABA concentration. A gating kinetic scheme for the alpha 1 beta 1 GABAA receptor channel was proposed that consisted of a single binding site for GABA and at least two open and five closed states. The kinetic properties of the alpha 1 beta 1 main conductance level differed from those of the spinal cord neuron (native) 27-pS main conductance level and the 19-pS subconductance level. The native main conductance and subconductance levels were characterized by longer openings and at least three open states. Based on the aforementioned observations, it appears that different subunit combinations produce receptor channels with different kinetic properties, but the basic mechanism of regulation by pentobarbital and picrotoxin may be similar for the different receptor channels. Also, it is unlikely that the 19-pS substate of the native GABAA receptor is produced by an alpha 1 beta 1 dimer.

Neurosteroid regulation of GABAA receptor single-channel kinetic properties of mouse spinal cord neurons in culture.

1. Single-channel kinetics of steroid enhancement of single gamma-aminobutyric acidA (GABA) receptor currents obtained from somata of mouse spinal cord neurones in culture were investigated using the excised outside-out patch-clamp recording technique. GABA (2 microM) and GABA (2 microM) plus androsterone (5 alpha-androstan-3 alpha-ol-17-one, AND, 10 nM-10 microM) or pregnanolone (5 beta-pregnan-3 alpha-ol-20-one, PRE, 100 nM-10 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage clamped at -75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were increased in the presence of the steroids. 2. GABA receptor currents were recorded with at least two conductance levels, a predominant or main-conductance level of about 28 pS (which contributed 96% of the current evoked) and a minor or sub-conductance level of about 20 pS. The current amplitudes of the two conductance levels were unchanged by the steroids. The gating (opening and closing) kinetics of both of the conductance levels were analysed. Findings for the main-conductance level are summarized below. 3. Both steroids increased the average GABA receptor channel open duration. Consistent with the increased GABA receptor channel average open duration, the steroids shifted frequency histograms of GABA receptor channel open durations to longer durations. Three exponential functions were required to fit best the frequency histograms of GABA open durations, consistent with at least three kinetic open states of the main-conductance level. Time constants obtained from the GABA receptor channel open-duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average GABA receptor channel open durations by the steroids was due to an increased relative proportion of the two longer open-duration time constants. The GABA receptor channel average open durations were increased by AND and PRE in a concentration-dependent manner by shifting the proportion of openings to the longer open time constants. At a concentration of 10 microM, the prolongation of the average open duration was decreased, suggesting that the GABA receptor channel was blocked by these steroids. 4. GABA receptor channel opening frequency was increased and average channel-closed duration was decreased by AND or PRE. Consistent with this, areas of the frequency histograms of channel closed durations were shifted to shorter durations. Closed frequency distributions were fitted best with five to six exponential functions, suggesting that the channel had multiple kinetic closed states. The three briefest time constants were not greatly altered by the steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic properties and regulation of GABAA receptor channels.

Single-channel recordings of GABAA receptor single-channel currents have been obtained from mouse spinal cord neurons in cell culture. Detailed kinetic analysis of single-channel main-conductance level currents has allowed development of a preliminary kinetic scheme which describes the gating of the GABAA receptor channel. The essential features of this kinetic scheme are presented in scheme 1 (see above). In this scheme, the GABAA receptor channel is envisioned to exist in multiple open and closed states. Properties can be broken into three main categories. First, the receptor can exist in a closed and nondesensitized set of states. In the kinetic model it is envisioned that there is an unbound (C13), a singly bound (C12), and two doubly bound (C11 and C10) closed states. The singly bound and doubly bound closed states are thought to open to three open states (O1, O2, O3). However, each of the open states opens to two distal closed states whose kinetic properties are similar for all three open states (C4-C9). Only one desensitized state (D0) has been incorporated into the model. While this characterization of desensitization is certainly incomplete, it is an initial step toward including the desensitization process which is clearly evident in whole-cell and single-channel recordings. This kinetic scheme should be considered only an initial working model. A number of features appear to be correct. First, all analyses of open time frequency histograms for GABA- and GABA agonist-induced single-channel openings have demonstrated the presence of at least three distinct open time constants. Furthermore, the concentration-dependent change in the relative frequency of occurrence of the three open states suggests that the open states occur from singly and doubly bound forms of the receptor. Second, the presence of two brief closed states adjacent to the open states appears fairly secure. However, it should be noted that the kinetic analysis primarily suggests that each open state opens to two brief adjacent closed states in a concentration-independent manner. While we have indicated that these two closed states are distal to the three open states, the actual assignment of the location of these states is unclear. Another interpretation of these data is that there is only a single distal closed state and that the proximal, extraburst closed states have very brief durations that are similar to each other. What appears clear, however, is that the open states can close to either brief closed state.(ABSTRACT TRUNCATED AT 400 WORDS)