Despite the introduction of over 15 third-generation anti-epileptic drugs, current medications fail to control seizures in 20-30% of patients. However, our understanding of the mechanisms mediating the development of epilepsy and the causes of drug resistance has grown substantially over the past decade, providing opportunities for the discovery and development of more efficacious anti-epileptic and anti-epileptogenic drugs. In this Review we discuss how previous preclinical models and clinical trial designs may have hampered the discovery of better treatments. We propose that future anti-epileptic drug development may be improved through a new joint endeavour between academia and the industry, through the identification and application of tools for new target-driven approaches, and through comparative preclinical proof-of-concept studies and innovative clinical trials designs.

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.

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.

SUMMARY1. The kinetic properties of the main conductance state of y-aminobutyric acidA (GABA) receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using patch clamp techniques.2. Whole-cell GABA receptor currents increased in a concentration-dependent manner from 0 5 to 5 /tM. R. L. MACDONALD, C. J. ROGERS AND R. E. TWYMAN suggesting that there was a population of single openings of short duration. The two longest burst time constants were longer than the two longest open time constants, suggesting that the bursts from the two longest burst components were composed of two or more openings.8. From these data we conclude that the main conductance of the GABA receptor channel has at least three open and two closed states whose time constants are concentration independent. The channel opens into a single-opening burst and into two complex bursts consisting of multiple openings. Single-opening bursts may be composed primarily of openings to the 01 state while complex bursts may be composed primarily of openings to the 02 and 03 states.9. We present a preliminary model for the gating of the main conductance of the GABA receptor channel.

The anticonvulsant activity of diazepam and phenobarbital may be mediated in part by enhancement of inhibition involving gamma-aminobutyric acid (GABA). While both diazepam and phenobarbital increase GABA receptor chloride current, they may have different mechanisms of action, since they bind to different sites on the GABA receptor-chloride channel complex. We used the patch clamp technique to compare the effects of diazepam and phenobarbital on single GABA receptor currents. Outside-out patches were obtained from mouse spinal cord neurons grown in cell culture for 2 to 4 weeks. GABA (2 microM) evoked single channel currents that occurred as single brief openings or in bursts of multiple openings. Diazepam (20 nM) and phenobarbital (500 microM) both increased the GABA receptor current by increasing mean open time without altering channel opening frequency. However, the temporal grouping of openings into bursts suggested that the enhancement occurred via different mechanisms. Diazepam increased the frequency of bursting GABA receptor currents with minimal effect on the duration of bursts. Phenobarbital increased the duration of bursting GABA receptor currents without altering the frequency of bursts. These results suggest that diazepam binds to a site that may enhance single channel burst frequency by increasing the affinity of GABA binding, while phenobarbital may stabilize the bursting open state of the channel by binding to a different modulatory site at or near the chloride channel.