We studied the role of desensitization at inhibitory synapses by comparing nonequilibrium GABAA channel gating with inhibitory postsynaptic currents (IPSCs). Currents activated by brief pulses of 1-10 mM GABA to outside-out patches from cultured hippocampal neurons mimicked GABA-mediated IPSCs. Although the average open time of single GABAA channels following brief pulses was less than 10 ms, channels entered long (tau = 38-69 ms) closed states and subsequently reopened. Movement through these states resulted in paired-pulse desensitization. The time required for deactivation after removal of agonist also increased in proportion to the extent of desensitization. These results suggest that visits to desensitized states buffer the channel in bound conformations and underlie the expression of long-lasting components of the IPSC. Reopening after GABAA receptor desensitization may thus enhance inhibitory synaptic transmission by prolonging the response to a brief synaptic GABA transient.

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Mutations in the GABAA receptor ␥2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a ␥2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of ␥2(R43Q) was seen in heterozygous mice in the absence of any change in ␣1 subunit surface expression, ruling out a dominant-negative effect. GABA Amediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.GABAA receptor ͉ genetics ͉ electroencephalogram ͉ trafficking ͉ synapse G ABA A receptors in the adult brain are important for inhibiting the activity of neurons in which they reside. Dysfunction of these receptors caused by familial mutations can give rise to febrile seizures (FS) and a variety of generalized epilepsy phenotypes (1-3). To date, five mutations have been reported in the GABA A ␥2 subunit gene with an array of seizure types seen in patients (1, 4-7). Childhood absence epilepsy (CAE) and FS were the main phenotypes in a large Australian family with an arginine to glutamine mutation at position 43 (R43Q) in the GABA A ␥2 subunit gene (1,8).Understanding how the GABA A ␥2(R43Q) mutation causes epilepsy is difficult. GABA A receptors themselves serve several roles. They regulate moment-to-moment brain function (9), play an important role in brain development (10), and have key roles in neuronal plasticity (11, 12) and response to brain injury (13-15). Epilepsy itself is a complex phenomenon involving the interaction of multiple cell types in networks within and between different brain regions that are likely to be influenced by GABA A receptor dysfunction caused by the R43Q mutation. Furthermore, in vitro analyses of the consequences of this mutation have shown inconsistent findings with a range of deficits in receptor pharmacology, trafficking, kinetics, or assembly (16-23) potentially implicated in disease pathogenesis.Clearly, the complex nature of epileptogenesis demands in vivo investigation. Genetic epilepsies provide a framework on which to investigate the consequences of causative mutations at a range of organizational levels within the brain, creating a chain of understanding from molecules to behavior. Linking this chain is impossible in humans because of the highly invasive methodology required and is severely limited in heterologous expression systems that lack necessary complexity. Mice mod...

At nicotinic and glutamatergic synapses, the duration of the postsynaptic response depends on the affinity of the receptor for transmitter (Colquhoun et al., 1977;Pan et al., 1993). Affinity is often thought to be determined by the ligand unbinding rate, whereas the binding rate is assumed to be diffusion-limited. In this view, the receptor selects for those ligands that form a stable complex on binding, but binding is uniformly fast and does not itself affect selectivity. We tested these assumptions for the GABAA receptor by dissecting the contributions of microscopic binding and unbinding kinetics for agonists of equal efficacy but of widely differing affinities. Agonist pulses applied to outside-out patches of cultured rat hippocampal neurons revealed that agonist unbinding rates could not account for affinity if diffusion-limited binding was assumed. However, direct measurement of the instantaneous competition between agonists and a competitive antagonist revealed that binding rates were orders of magnitude slower than expected for free diffusion, being more steeply correlated with affinity than were the unbinding rates. The deviation from diffusion-limited binding indicates that a ligand-specific energy barrier between the unbound and bound states determines GABAA receptor selectivity. This barrier and our kinetic observations can be quantitatively modeled by requiring the participation of movable elements within a flexible GABA binding site.