GABAB receptor, localization and regulation

GABAB receptors are G-protein coupled receptors for gamma-amino butyric acid, the main inhibitory neurotransmitter in the brain. Functional GABAB receptors are obligate heterodimers composed of GABAB1 and GABAB2 subunits. The GABAB1 subunit exists in two isoforms, GABAB1a and GABAB1b, that can be differentiated by a pair of sushi domains exclusively located on the ectodomain of GABAB1a. As a consequence, two distinct receptor subtypes, GABAB(1a,2) and GABAB(1b,2), are present in the brain. Depending on their subcellular localization, GABAB receptors exert distinct regulatory effects on synaptic transmission. Presynaptically, GABAB receptors inhibit Ca2+ influx by closing voltage-gated Ca2+-channels therefore regulating neurotransmitter release. Postsynaptically, GABAB receptors activate inwardly rectifying Kir3-type K+-channels leading to hyperpolarisation of the postsynaptic membrane. Recently, it has become clear that GABAB(1a,2) and GABAB(1b,2) receptors convey individual functions, which are, at least in part, related to their distinct subcellular distribution. The aim of this thesis was to gain further insight into the function of GABAB receptors by characterizing their localization at the ultrastructural level in respect to effector channels and subtype composition. Moreover, it was of interest to study the dynamic regulation of GABAB receptors in response to synaptic activity. In the first part of this thesis, the localization of GABAB receptors and Kir3-type effector channels was investigated in the CA1 region of the hippocampus. It could be demonstrated that postsynaptic GABAB receptors colocalize with the Kir3.2 subunit of K+-channels in dendritic spines, but not in dendritic shafts of CA1 pyramidal cells (chapter 6.1.; Kulik et al., 2006). The differential distribution of GABAB1 subunit isoforms at the mossy fiber-CA3 pyramidal neuron synapse was investigated in the second part of this work. Due to the lack of isoform specific antibodies, mice selectively expressing GABAB1a or GABAB1b were used. It could be shown that mainly the GABAB1a subunit isoform contributes to the composition of presynaptic GABAB receptors whereas GABAB1b is the predominant GABAB1 subunit isoform on the postsynaptic side. Electrophysiological recordings were used to assess the contribution of the two different GABAB1 subunit isoforms to functional pre- and postsynaptic receptors in response to pharmacological as well as physiological GABAB receptor activation. The findings illustrate that the spatial segregation of GABAB1 subunit isoforms at mossy fiber terminals is sufficient to produce a strictly subtype–specific response (chapter 6.2.; Guetg et al., 2009). In the third part of this work, a new mouse model containing a GABAB1-eGFP transgene, allowing the visualization of GABAB receptors, was generated. Crossing the GABAB1-eGFP transgene into the GABAB1 deficient background allowed the study of GABAB receptors tagged with a fluorescent protein under expression of endogenous promoter elements. Therefore these mice provide a useful tool to visualize the spatio-temporal distribution of GABAB receptors in vivo and in vitro (chapter 6.3.; Casanova et al., 2009). The dynamic regulation of surface GABAB receptors induced by glutamate was investigated in primary hippocampal neurons and the results are presented in the last part of this thesis. Activation of NMDA receptors resulted in a decrease of surface GABAB receptor levels. This decrease involved Ca2+-dependent activation of CaMKII. A CaMKII phosphorylation site within the cytoplasmic domain of the GABAB1 subunit was identified. Evidence that phosphorylation of this site is essential for the observed effect of NMDA receptor activation on GABAB surface receptors is presented in this thesis. In conclusion, it could be demonstrated that GABAB receptors are dynamically regulated and interact with other receptors and kinases. The results obtained, implicate that activity-dependent regulation of GABAB receptors is potentially involved in the modulation of synaptic strength (chapter 6.4.).