Abstracts

RATIONALE: An arginine to glutamine mutation at position 43 within the GABA [gamma]2 subunit has been identified in patients with childhood absence epilepsy and febrile seizures¹. Our objective was to explore the possibility that the [gamma]2(R43Q) mutation contributes to epileptogenesis via alterations in the kinetics of inhibitory postsynaptic currents (IPSCs). We mimicked synaptic receptor activation by applying brief pulses of GABA to outside-out patches containing wild type [alpha]1[beta]2[gamma]2 or [alpha]1[beta]2[gamma]2(R43Q) receptors.
METHODS: Recordings were made from outside out patches excised from HEK-293 cells that were transiently transfected with recombinant [alpha]1, [beta]2, and [gamma]2 subunits. Patches were voltage clamped at -60 mV and placed in the stream of a multi-barrel flowpipe array mounted on a piezo-electric bimorph. Solution exchange times were typically [lt] 200 [mu]s.
RESULTS: After a 2 ms pulse of 10 mM GABA, currents from mutant receptors deactivated significantly more slowly than currents from wild type (wt) receptors. Weighted time constants determined from multi-exponential fits to the time courses of deactivation ([tau][sub]w[/sub]) were 46 [plusminus] 12 ms (wt) and 98 [plusminus] 12 ms (mutant, mean [plusminus] sem) When mutant receptors were presented with a 500 ms step of saturating GABA slowed deactivation was also accompanied by a markedly increased fast component of desensitization ([tau][sub]w[/sub]-wt = 371 [plusminus] 31 ms, [tau][sub]w[/sub]-mutant = 74 [plusminus] 12 ms). Paired-pulse experiments demonstrated that increased fast desensitization of [alpha]1[beta]2[gamma]2(R43Q) receptors caused a profound increase in paired-pulse depression from which the receptors were slow to recover. Currents from patches containing both wild type and mutant receptors were relatively insensitive to block by 10 [mu]M zinc confirming that the [gamma] subunit was efficiently expressed in both instances.
CONCLUSIONS: During inhibitory synaptic transmission, the slow deactivation caused by the [gamma]2(R43Q) mutation predicts that IPSCs would be prolonged. However, the mutation also greatly increases the rate and extent of desensitization, and dramatically slows the recovery from desensitization following a brief GABA pulse. Therefore, rapid and repetitive stimulation of inhibitory synapses may cause [alpha]1[beta]2[gamma]2(R43Q) receptors to accumulate in desensitized states, resulting in a progressive loss of inhibition and possibly leading to epileptiform activity.
¹ Wallace RH, Marini C, Petrou S, Harkin LA, Bowser DN, Panchal RG, Williams DA, Sutherland GR, Mulley JC, Scheffer IE and Berkovic SF. Mutant GABA(A) receptor gamma2-subunit in childhood absence epilepsy and febrile seizures. Nat Genet 28: 49-52., 2001.
[Supported by: grants from the Epilepsy Foundation (DW and MVJ), the National Health and Medical Research Council of Australia and Bionomics Ltd.]