Optoelectrical study of neuronal calcium nanodomains

Abstract

In neurons, coupling between calcium influx and membrane voltage in pre- synaptic terminal is essential for neurotransmitter release, hyperpolarization and re- polarization and shaping of Ca2+ dendritic spikes. In the functional context, these processes are fine-tuned and controlled by complexes called “nanodomains”, which are formed by the tight association between calcium permeable channels and large conductance voltage- and calcium-gated potassium channels (BK). The structural mechanisms involved in the Ca2+ dependent activation of BK channels and deducing how these Ca2+ signals are integrated and converted into an outflux of potassium ions are intriguing questions that have to be comprehensively studied.

During this PhD, we aimed to advance our knowledge about the precise func- tion of BK channels within the nanodomains as well as its structural roles in forming the complexes with N-methyl-D-aspartate glutamate receptors (NMDARs). In order to fulfil our aims, we used a combination of the most recent techniques developed in the field, including unnatural amino acids, self-labelling enzymes, superresolution microscopy and single molecule pull-down. This allowed us to study the specific structural rearrangements involved in the activation of this channel by Ca2+, as well as to develop tools to study the protein-protein interactions between BK channel and NMDARs.

We demonstrated the existence of an intrasubunit bridge between the Ca2+ binding sites of BK channel as well as the crucial role of the intersubunit interfaces in the activation of the channel by this divalent cation. We reconstituted BK channel-NMDARs complexes in heterologous systems and studied the influence of GluN2 NMDAR composition in BK channel activation. Additionally, we constructed functional fusion proteins between self-labelling enzymes and BK channel, or NMDARs, and validated them under different microscopy approaches.