title: Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies.
abstract: The ClC-2 Cl- channel has been postulated to play a role in the inhibitory GABA response in neurons or to participate in astrocyte-dependent extracellular electrolyte homeostasis. Three different mutations in the CLCN2 gene, encoding the voltage-dependent homodimeric ClC-2 channel, have been associated with idiopathic generalized epilepsy (IGE). We study their function in vitro by patch clamp and confocal microscopy in transiently transfected HEK-293 cells. A first mutation predicts a premature stop codon (M200fsX231). An altered splicing, due to an 11-bp deletion in intron 2 (IVS2-14del11), predicts exon 3 skipping (Delta74-117). A third is a missense mutation (G715E). M200fsX231 and Delta74-117 are nonfunctional and do not affect the function of the normal (wild type, WT) channel. Neither M200fsX231 nor Delta74-117 reach the plasma membrane. Concerning the IVS2-14del11 mutation, we find no difference in the proportion of exon-skipped to normally spliced mRNA using a minigene approach and, on this basis, predict no alteration in channel expression in affected individuals. G715E has voltage dependence and intracellular Cl- dependence indistinguishable from WT channels. ClC-2 channels are shown to be sensitive to intracellular replacement of ATP by AMP, which accelerates the opening and closing kinetics. This effect is diminished in the G715E mutant and not significant in WT+G715E coexpression. We do not know whether, in a situation of cellular ATP depletion, this might become pathological in individuals carrying the mutation. We postulate that loss of function mutation M200fsX231 of ClC-2 might contribute to the IGE phenotype through a haploinsufficiency mechanism.
authors.affiliation: Centro de Estudios Científicos, Valdivia, Chile.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Niemeyer
authors.forename: Yamil R
authors.initials: YR
authors.lastname: Yusef
authors.forename: Isabel
authors.initials: I
authors.lastname: Cornejo
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Flores
authors.forename: Francisco V
authors.initials: FV
authors.lastname: Sepúlveda
authors.forename: L Pablo
authors.initials: LP
authors.lastname: Cid
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D017398:Alternative Splicing / Q000235:genetics; D000090007:CLC-2 Chloride Channels; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics* / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D018388:Codon, Terminator / Q000235:genetics; D004594:Electrophysiology; D004829:Epilepsy, Generalized / Q000235:genetics*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D009154:Mutation / Q000235:genetics*; D021381:Protein Transport
chemical_list: D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D018388:Codon, Terminator; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate