%0 Generic
%A Schumacher, Stefanie Birgitta
%D 2010
%F heidok:10920
%K KCC2 , Glyzin RezeptorKCC2 , glycine receptor , spinal neurons
%R 10.11588/heidok.00010920
%T KCC2 plays a crucial role during the maturation of spinal neurons by regulating the expression of the GlyR alpha subunit and gephyrin
%U https://archiv.ub.uni-heidelberg.de/volltextserver/10920/
%X In spinal cord and brain stem, the GlyR is the major receptor type to mediate inhibitory synaptic transmission. Together with the GlyRb subunit, the GlyRa1 subunit forms the adult form of the receptor. Activation of the pentameric GlyR by glycine results in an increased permeability of the GlyR channel for chloride. In juvenile stages, the cell is depolarized whereas adult neurons experience a hyperpolarization. Not least, the activity of KCC2, changing the intracellular chloride level from a high to a low adult state, contributes considerably to the maturation of the neurons. In this thesis, the approximate time point of the switch to occur in vitro could be determined to div 14 via mRNA expression studies of the distinct GlyR subunits. The obtained mRNA data also indicate that the GlyRb subunit exhibits the highest RNA expression whereas GlyRa1 expression is even lower than that of GlyRa2. Taken together, the results suggest that adult neurons as well as juvenile neurons still express heteromeric α2β and/or homomeric α2 receptors. Strychnine application to the SN cultures resulted in a down regulation of KCC2 expression in a dose-dependent manner at div 14. This finding indicates that the activity of the GlyR may regulate the expression of KCC2 to some extent. Subsequent treatments with Ca2+ channel blockers and Ca2+ chelators revealed a role for Ca2+ to destabilize the KCC2 complex. Although suppression of the entire synaptic transmission in the culture by TTX application as well as catching all free Ca2+ ions also reduce KCC2 expression, the latter seems not to be dependent on Ca2+ influx via L-type channels. However, KCC2 expression either depends on Ca2+ being present within the cell or Ca2+ influx through another type of Ca-channel than L-type. To clarify the role of KCC2 in the GlyR subunit switch, its expression successfully was down regulated upon transduction by a silencing shRNA construct. Immunoblot and localization of immunoreactivities reveal a decrease in the expression of the adult GlyRa1 subunit following KCC2 knockdown. The expression of gephyrin is affected as well. In the main, no additionally investigated synaptic proteins are concerned. Therefore, the neurons are supposed to remain in a juvenile state in terms of the GlyR when KCC2 is not expressed properly. The question arising is, whether the pure presence or the activity of KCC2 is responsible for the correct GlyRa1 expression. It also remains unclear whether knockdown of KCC2 affects GlyRa1 expression and hence gephyrin expression, the other way around or whether even both proteins directly are affected by loss of KCC2. I suggest that loss of KCC2 impairs the expression of the scaffolding protein gephyrin via unknown cytoskeletal mechanisms or signalling pathways. The absence of gephyrin in turn directs loss of the adult GlyR consisting of GlyRa1 and GlyRb. This loss might result from either down regulation of GlyRa1 expression or removal of the adult receptor from the membrane and subsequent degradation.