Altered Kinetics and Benzodiazepine Sensitivity of a GABAA Receptor Subunit Mutation [␥2(R43Q)] Found in Human Epilepsy

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [␥2(R43Q)] found in human epilepsy David N. Bowser*†, David A. Wagner†‡, Cynthia Czajkowski‡, Brett A. Cromer§, Michael W. Parker§, Robyn H. Wallace¶, Louise A. Harkin¶, John C. Mulley¶, Carla Mariniʈ, Samuel F. Berkovicʈ, David A. Williams*, Mathew V. Jones‡, and Steven Petrou*,** *Department of Physiology, University of Melbourne, Victoria 3010, Australia; ‡Department of Physiology, University of Wisconsin, Madison, WI 53706; §Biota Structural Biology Laboratory, St. Vincent’s Institute for Medical Research, Fitzroy, Victoria 3065, Australia; ¶Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital, Adelaide, SA 5006, Australia; and ʈEpilepsy Research Institute and Department of Medicine (Neurology), University of Melbourne, and Austin and Repatriation Medical Centre, Heidelberg, Victoria 3081, Australia Edited by Bert Sakmann, Max Planck Institute for Medical Research, Heidelberg, Germany, and approved September 3, 2002 (received for review May 28, 2002) ␥ The -aminobutyric acid type A (GABAA) receptor mediates fast GABAA receptor is a heterooligomeric complex of five subunits. inhibitory synaptic transmission in the CNS. Dysfunction of the Each subunit has a large, N-terminal extracellular domain, four GABAA receptor would be expected to cause neuronal hyperexcit- transmembrane domains, and a small, extracellular C-terminal ability, a phenomenon linked with epileptogenesis. We have tail. Multiple subunits have been cloned and divided into families investigated the functional consequences of an arginine-to- based on their sequence homology. The subunits also are ␥ glutamine mutation at position 43 within the GABAA 2-subunit differentially expressed in various brain regions and confer found in a family with childhood absence epilepsy and febrile distinct pharmacology to the expressed GABAA receptor (15, seizures. Rapid-application experiments performed on receptors 16). Most native receptors contain ␣-, ␤-, and ␥-subunits, with expressed in HEK-293 cells demonstrated that the mutation slows ␣ ␤ ␥ 1 2 2 being the most common combination (17). The receptor GABAA receptor deactivation and increases the rate of desensiti- has two GABA-binding sites formed by the extracellular do- zation, resulting in an accumulation of desensitized receptors mains at the interface of ␣␤-subunits. The receptor also is during repeated, short applications. In Xenopus laevis oocytes, modulated allosterically by benzodiazepines, believed to bind at two-electrode voltage-clamp analysis of steady-state currents ob- the interface of ␣␥-subunits. Binding of benzodiazepines poten- ␣ ␤ ␥ ␣ ␤ ␥ tained from 1 2 2 or 1 2 2(R43Q) receptors did not reveal any tiates GABA-activated ClϪ currents but, alone, is unable to differences in GABA sensitivity. However, differences in the ben- activate the receptor. The exact mechanism of receptor modu- zodiazepine pharmacology of mutant receptors were apparent. lation by benzodiazepines remains unclear. Mutant receptors expressed in oocytes displayed reduced sensi- Mutations in GABA receptor subunit genes recently have tivity to diazepam and flunitrazepam but not the imidazopyridine A been described in human epilepsy (18–20). Two families with the zolpidem. These results provide evidence of impaired GABA A GEFSϩ phenotype had different mutations in the ␥ -subunit receptor function that could decrease the efficacy of transmission 2 gene, severely reducing or abolishing ClϪ currents in response to at inhibitory synapses, possibly generating a hyperexcitable neu- GABA in Xenopus laevis oocytes. A French family (20) had a ronal state in thalamocortical networks of epileptic patients pos- sessing the mutant subunit. K289M mutation that resulted in an amino acid change in the extracellular loop between the second and third transmembrane ͉ ͉ domains. We described an Australian family with a nonsense rapid agonist application two-electrode voltage clamp Xenopus oocytes ␥ mutation that caused truncation of the 2-subunit at position 351 between the third and forth transmembrane domains (19). pilepsy, one of the most common neurological disorders, is Heteromeric GABA receptor complexes containing GFP- Echaracterized by episodic seizures that are caused by parox- ␥ tagged truncated 2-subunits were found to be trapped within ysmal, synchronized discharges of hyperexcitable neuronal pop- the endoplasmic reticulum, thereby inhibiting expression at the ulations. Many human epileptic syndromes have a genetic com- cell membrane. Different phenotypes of simple febrile seizures, ponent, and the molecular basis of a few inherited epilepsies is childhood absence epilepsy and GEFSϩ, were observed in a now known (1). Most genes implicated in epilepsy to date code large Australian family with a missense mutation in the extra- for ion channel subunits, both ligand-gated and voltage-gated cellular domain of the ␥ -subunit (18). The mutation (R43Q) (2). For example, mutations within the ␣- and ␤-subunits of the 2 resulted in the replacement of a highly conserved arginine, found voltage-gated sodium channel family have been found in families in most ligand-gated receptor subunits, for a glutamine at with generalized epilepsy with febrile seizures plus (GEFSϩ; position 43. Although expression of this mutant subunit in refs. 3 and 4). Mutations within the voltage-gated potassium Ϫ channel subunits KCNQ2 and KCNQ3 cause the benign familial X. laevis oocytes did not alter GABA-induced Cl currents, lack neonatal convulsions phenotype (5–9), whereas mutations in the of potentiation of such currents with low benzodiazepine con- acetylcholine ligand-gated receptor family are responsible for centrations was observed. familial nocturnal frontal lobe epilepsy (10–14). In vitro inves- tigations with the oocyte expression system and two-electrode This paper was submitted directly (Track II) to the PNAS office. voltage-clamp assay generally suggest that these mutations Abbreviations: GABAA, ␥-aminobutyric acid type A (receptor); GEFSϩ, generalized epilepsy would result in neuronal hyperexcitability. with febrile seizures plus; HEK, human embryonic kidney. ␥ The -aminobutyric acid type A (GABAA) receptor is the † Ϫ D.N.B. and D.A.W. contributed equally to this work. predominant ligand-gated Cl ion channel conferring fast in- **To whom correspondence should be addressed at: Laboratory of Biophysics and Molec- hibitory synaptic transmission in the CNS and, therefore, is a ular Physiology, Department of Physiology, University of Melbourne, Victoria 3010, prime candidate for involvement in epileptogenesis. The Australia. E-mail: [email protected]. 15170–15175 ͉ PNAS ͉ November 12, 2002 ͉ vol. 99 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.212320199 Downloaded by guest on September 29, 2021 Impaired sensitivity to benzodiazepines may explain some Expression in HEK-293 Cells and Outside-Out Patch-Clamp Assay. aspects of the epilepsy phenotypes observed. Here, we postulate HEK-293 cells were cultured in minimum essential medium with ␥ that the 2(R43Q) mutation may have additional and significant Earle’s salts (Life Technologies) containing 10% FBS (Sigma) in pathophysiological effects. This is due, in part, to the observation a37°C incubator under a 5% CO2 atmosphere. When the cells that R43 is so highly conserved amongst other ligand-gated reached 60–90% confluence in 60-mm culture dishes, they were ␮ ␣ ␤ ␥ channels that do not interact with benzodiazepines that it may transfected with 1 g each of 1, 2, and 2 (WT or R43Q) play a more fundamental role in receptor function. We now have cDNAs subcloned into the pcDNA3.1(ϩ) vector and 200 ng of examined both GABA sensitivity and responses to benzodiaz- a vector containing the T cell membrane antigen CD8 cDNA epines by using two-electrode voltage-clamp recordings of (gift from Robert Coronado, Univ. of Wisconsin, Madison, WI) Xenopus oocytes heterologously expressing WT and mutation by using the Lipofectamine 2000 reagent (GIBCO) according to GABA receptors. The oocyte system, however, has relatively the supplied protocol. Twenty to 28 h posttransfection, the cells slow temporal resolution, whereas during synaptic transmission, were replated on 12-mm sterile glass coverslips, transferred to a receptors are activated by pulses of neurotransmitter that reach 30°C incubator, and recorded at 24–96 h postreplating. Trans- millimolar concentrations and decay within a millisecond (21). fected cells were recognized by incubation with anti-CD8 anti- To obtain sufficient temporal resolution, we used rapid-solution body beads (Dynal, Oslo). switches on outside-out patches from human embryonic kidney Recordings from outside-out patches excised from HEK cells (HEK-293) cells to approximate the conditions at synapses. We were made by using borosilicate glass pipettes filled with 140 mM ␥ ͞ ͞ ͞ found that the 2(R43Q) mutation slows receptor deactivation KCl 10 mM EGTA 2mMMgATP20 mM phosphocre- and increases fast desensitization. These kinetic differences atine͞10 mM Hepes, pH 7.3. Patches were voltage-clamped at could lead to reduced inhibition during periods of high activity Ϫ60 mV and placed in the stream of a multibarreled flowpipe and, thus, contribute to the epileptic phenotypes. array (Vitro Dynamics, Rockaway, NY) mounted on a piezo- electric bimorph (Vernitron, Bedford, OH). A computer- Materials and Methods controlled constant current source drove the bimorph to move Construction of

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