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Dynamic interplay between voltage-gated calcium channels and presynaptic cytomatrix

Subject Area Molecular Biology and Physiology of Neurons and Glial Cells
Term from 2020 to 2025
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 436933646
 
Final Report Year 2025

Final Report Abstract

The research project investigated the organisation and function of glutamatergic synapses. The main aim was to investigate the molecular mechanisms in which the cytomatrix proteins Bsn and Pclo are involved and how they interact with voltage-gated calcium channels in the presynaptic membrane and influence their function. Previous work has suggested that Bsn controls the recruitment of synaptic vesicles (SV) and defines the density of voltage-gated calcium channels (VGCC) in the active zone. The aim of this project was to investigate to what extent the functions of Pclo are homologous or exclusive. Different mouse models were used and evaluated to manipulate the expression of Pclo, Bsn and Cav2.1 channels and to investigate the effects on synaptic transmission. Bsn was shown to significantly modulate the population of release-competent SV via the CDK5/calcineurin and cAMP/PKA signalling pathways. In characterising a new conditional knock-out model for Pclo, we found a shift in the equilibrium of synaptic VGCC populations. In parallel to this work on the scaffold proteins, we analysed the function of different Cav2.1 splice variants for synaptic transmission. Here we characterised the C-terminal splice variants of exon 37 and exon 47. Exon47 mediates the interactions of Cav2.1 with the cytomatrix proteins RIM, RBP and indirectly also with Bsn/Pclo. Functional studies showed that exon 47 has no effect on the synaptic recruitment of the channels, but has a decisive influence on the release properties of the presynapse. Measurements of the lateral mobility of Cav2.1 channels in the presynaptic membrane show that the long splice variant of the Cav2.1 channel is stabilised by the absence of Bsn, which could not be observed with the short splice variant (without exon 47). These observations therefore suggest a possible role for the alternative splicing of exon 47 in the regulation of transmitter release. No differences in expression of Cav2.1 channels with and without exon 47 could be detected in different populations of glutamatergic neurones, but it remains to be clarified whether there is an activity- or stimulus-dependent regulation. We also investigated the extent to which Bsn and Pclo contribute to the organisation of phase-separated complexes at the active zone and thus modulate the transmission properties of the synapse. In a parallel study in the NMJ of the fly Drosophila melanogaster, we were able to confirm the hypothesis that even small changes in the arrangement of the calcium channels are effective and also depend on indirect interactions with scaffold proteins (Bruchpilot). This often-assumed precise arrangement of the calcium channels in relation to the SV does not appear to be realised at the molecular level via strong protein-protein bonds, but is based on a very dynamic interaction of the proteins. In future projects, we will further investigate this hypothesis and mechanistically analyse the influence of the identified signalling pathways, such as CDK5, calcineurin or cAMP/PKA.

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