Der spannungsgesteuerte Chloridkanal CIC-2 und seine Bedeutung in der Pathogenese von idiopathischer generalisierter Epilepsie

ClC-2 is a member of the ClC-family of voltage-gated chloride channels. Within this family, ClC-2 is unique in being activated by hyperpolarization, low extracellular pH and hypotonic cell swelling. In this study, several biophysical properties of the human ClC-2 channel have been functionally characterized for the first time and compared with the corresponding properties of the rat isoform. Both proteins were expressed heterologously in tsA201-cells and were analyzed electrophysiologically using the whole-cell patch-clamp technique. hClC-2 and rClC-2 hardly differ in their activation properties. Both channels are virtually closed at positive potentials and activate slowly upon hyperpolarization. However, the human ClC-2 displays slower activation and deactivation kinetics than the rat isoform. A formerly hypothesized function as an inactivation domain for the cytoplasmatic N-terminus of ClC-2 could not be verified. A corresponding N-terminal deletion mutant exhibits a nearly unchanged gating behaviour which argues against the proposed inactivation model for this channel. However, the changed kinetics of the mutant imply an important role of the N-terminus in gating of ClC-2. Furthermore, it could be shown that the current block by external iodide is dependent on the membrane potential as well as on the conformational state of the channel. This argues against the existence of an intracellular inactivation domain as well. A dependence of the activation behaviour of ClC-2 on the transmembrane chloride gradient could be proven: the channel activates only at potentials negative to the reversal potential and therefore constitutes an exclusive efflux pathway for chloride ions. Based on this property, a model for the role of ClC-2 in neuronal chloride homeostasis has been proposed assigning the channel an important function in establishing and maintaining a low intracellular chloride concentration. In the second part of this work, three mutations of ClC-2 were studied that have recently been identified in patients with idiopathic generalized epilepsy (IGE). Two of these mutants, M200fsX231 and del74-117, were non-functional, neither as homodimers nor as heterodimers with one wildtype subunit. A correct trafficking of the mutated proteins to the cell membrane was demonstrated by fluorometric experiments and could therefore be discarded as possible cause for the dominant-negative effect of these mutations. Whether the lack of functionality is due to the missing ability of the mutated subunits to interact with wildtype subunits could not be shown unambiguously in FRET experiments. The loss of ClC-2 function in patients carrying these mutations might lead to an intracellular chloride accumulation that could result in an excitatory GABAergic response and neuronal hyperexcitability and thus represent a possible explanation for the epileptic phenotype. The third IGE mutation, G715E, does not impair the functionality of ClC-2 but leads to an altered voltage-dependence of channel gating: mutated channels open already at less negative potentials than the wildtype isoform. Under certain conditions, for example during the repolarization phase after intense synaptic activity, they conduct an increased outward chlorid current possibly leading to recurrent membrane depolarization and repeated action potentials. The G715E mutation therefore seems to cause epileptic seizures by a distinct mechanism comprising a gain-of-function of ClC-2. The results of this thesis demonstrate the importance of gating in ClC-2 for its function and establish CLCN2 as a disease-causing gene in idiopathic epilepsies.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2004

Author dc:creator

• Warnstedt, Maike Ursula

Contributors dc:contributor

• Fahlke, Christoph

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• ClC-2
• Chloridkanal
• Aktivierung
• transmembraner Chlorid-Gradient
• Epilepsie-Mutationen

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-121383%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:59613