On the functional consequences of epilepsy-causing mutations located in ion channels and the role of cytoplasmic protein regions in fast and slow inactivation of voltage-gated sodium channels

Ion channels provide the basis for excitability in nerve and muscle cells. This thesis presents the functional characterization of K+ and Na+ channel mutations causing inherited epilepsies and a structure-function study about the role of two cytoplasmic protein regions of the voltage-gated Na+ channel. Three epilepsy causing mutations 2513delG in the KCNQ2 channel (causing benign familial neonatal convulsions) and T685M and R1460H in the voltage-gated Na+ channel (associated with generalized epilepsy with febrile seizures plus) were expressed and functionally characterized. For all three mutations changes were found explaining the occurrence of epileptic seizures. Fast inactivation in voltage-gated Na+ channels is believed to function in the so-called "hinged-lid" fashion - a hydrophobic particle of three amino acids (IFM) occludes the pore from the intracellular site of the membrane. Possible binding sites for the inactivation particle are the D4/S6 segment and the D4/S4-S5 interloop. Two mutations in the intracellular loop D4/S4-S5 (L1482C/A) were investigated. Both mutations introduced prominent effects on fast and slow inactivation, demonstrating that D4/S4-S5 loop is involved in the regulation of the before mentioned processes. The applied thermodynamic analysis showed no functional cooperativity of D4/S4-S5 region and the inactivation particle in fast inactivation. To investigate in detail the role of segment D4/S6 in Na+ channel gating, the amino acids at positions F1586, V1589, M1592 and I1596 were substituted by cysteines and the effects of the mutations and application of MTS reagents, covalently binding to cysteines, were studied. All gating transitions, following activation were strongly affected, demonstrating a central functional role of segment D4/S6 in the gating of voltage-dependent Na+ channels. Additionally, the reported effects propose that the slow inactivation gate in Na+ channels contains the cytoplasmic part of segment D4/S6

Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1-glucosylation kinetics are decisive for cell fate.

Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species-mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC-5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC-5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered