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NANOPDICS Report Summary

Project ID: 648936
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - NANOPDICS (Optoelectrical Dynamics of Ion channel Activation in Calcium Nanodomains)

Reporting period: 2015-09-01 to 2017-02-28

Summary of the context and overall objectives of the project

In neurons, sites of Ca2+ influx and Ca2+ sensors are located within 20-50 nm, in subcellular “Ca2+ nanodomains”. Such tight coupling is crucial for the functional properties of synapses and neuronal excitability. Two key players act together in nanodomains, coupling Ca2+ signal to membrane potential: the voltage-dependent Ca2+ channels (VDCC) and the large conductance Ca2+ and voltage-gated K+ channels (BK). BK channels are characterized by synergistic activation by Ca2+ and membrane depolarization, but the complex molecular mechanism underlying channel function is not adequately understood. Information about the pore region, voltage sensing domain or isolated intracellular domains has been obtained separately using electrophysiology, biochemistry and crystallography. Nevertheless, the specialized behavior of this channel must be studied in the whole protein complex at the membrane in order to determine the complete range of structures and movements critical to its in vivo function. Using a combination of genetics, electrophysiology and spectroscopy, our group has measured for the first time structural rearrangements accompanying whole BK channel activation at the membrane. From this unique position, our first goal is to fully determine the real time structural dynamics underlying the molecular coupling of Ca2+, voltage and activation of BK channels in the membrane environment, its regulation by accessory subunits and channel effectors. BK subcellular localization and role in Ca2+ nanodomains make these channels perfect candidates as reporters of local changes in [Ca2+] restricted to specific nanodomains close to the neuronal membrane. In our laboratory we have created fluorescent variants of the channel that report BK activity induced by Ca2+ binding, or Ca2+ binding and voltage. Our second aim in this proposal is to optimize and deploy these novel optoelectrical reporters to study physiologically relevant Ca2+-induced processes both in cellular and animal models.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Until now, the NANOPDICS Project has successfully set up the NANOPDICS-MOLCAN Lab, at the School of Medicine of the Universidad de La Laguna, next to the new building of the CIBICAN. This lab includes a molecular biology and biochemistry laboratory, an electrophysiology laboratory (including two dark rooms where the microscopes are installed), office space for the PI, postdocs and students, and a small kitchen. The NANOPDICS team is now complete and fully functional and consists of the PI, 3 postdocs, 1 PhD students (and a second who will start in May 2017), 1 technician and a Lab Manager. A Project website is ready. Preliminary investigations have been implemented and the main achievements include the characterization of new sites proposed to be involved in BK modulation by Calcium, the identification of independent movements triggered by specific activation of ion binding sites in the BK gating ring (Miranda et al, PNAS 2016), preliminary super-resolution images of Cav-BK complexes in heterologous expression systems, Proximity ligand assay experiments characterizing Cav-BK complexes composed of various Cav types, unnatural aminoacids insertion into BK channels and successful functional assays, development of potential new sensors after insertion of circularly permutated GFP into BK channel proteins. In addition, we have started the generation of a transgenic mouse where the GFP is inserted in the 667 site into the endogenous BK channels. The new lab and project has been presented at different international academic institutions, 4 renowned researchers have visited the facilities and imparted specialized seminars for the project. The PI has organized a workshop within the context of the Biophysical Society Annual Meeting, which was related to the topic of this project (this workshop was not funded by the ERC project).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

At the moment this summary is elaborated, the project has achieved a unique lab setup that will allow to perform electrophysiological recordings from oocytes, cell lines, neuron cultures and brain slices preparations. This recordings can be simultaneourly performed using fluorescence approaches, including superresolution (STORM). We are in the process of generating novel transgenic mouse lines using the CRISPR-Cas9 technique. In its current state, the lab facilities can provide a service to the scientific community at large.

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