Alteration of GABAA Receptor Function Following Gene Transfer of the CLC-2 Chloride Channel

Abstract

The effect of GABAA receptor activation varies from inhibition to excitation depending on the state of the transmembrane anionic concentration gradient (delta anion). delta anion was genetically altered in cultured dorsal root ganglion neurons via adenoviral vector-mediated expression of ClC-2, a Cl- channel postulated to regulate the Cl- concentration in neurons in which GABAA receptor activation is predominantly inhibitory. ClC-2 expression was verified by the presence of the appropriate mRNA, protein, and membrane conductance. CIC-2 expression resulted in a large negative shift in the Cl- equilibrium potential (ECl) that attenuated the GABA-mediated membrane depolarization and prevented GABAA receptor-mediated action potentials. These results establish that gene transfer of transmembrane ion channels to neurons can be used to demonstrate their physiological function, and that delta anion can be genetically manipulated to alter the function of neuronal GABAA receptors in situ.

Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies

“…All affected family members were found to be heterozygous with respect to the mutation G2144A (G715E). [13][14][15] . We sequenced all 24 exons and adjacent splice sites of CLCN2 in two affected individuals from 46 unrelated families with IGE.…”

Perturbed Chloride Homeostasis and GABAergic Signaling in Human Temporal Lobe Epilepsy

“…For instance, the depolarizing shift in GABA reversal potential induced by deafferentation of the colliculus has been attributed to posttranslational modifications rather than reduced KCC2 expression (Vale et al, 2003). Third, other molecules that participate in Cl Ϫ homeostasis (Staley et al, 1996;Payne et al, 2003;Farrant and Kaila, 2007) may contribute to the shift in GABAergic signaling associated with interictal rhythmicity in human epilepsy.…”

Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies

“…This point can only be speculated upon, given the fact that the function of normal ClC-2 in the brain has not been elucidated. In neurons ClC-2 is postulated to prevent paradoxical GABA A R-mediated excitatory actions (42), while a role in extracellular electrolyte homeostasis is a proposed function in astrocytes (38). A dysfunction of either of these processes could lead to disturbances in neuronal excitability and could explain the epileptic phenotype.…”

“…In these two cell types the channel might play different roles. In neurons, it is speculated that because of its activation by intracellular Cl Ϫ , ClC-2 would prevent accumulation of this anion above equilibrium (41,42). Intracellular accumulation of Cl Ϫ might take place during high-frequency stimulation due to an increase in extracellular K ϩ with reversal of KCl cotransport (7,23).…”