Report

Teaser, summary, work performed and final results

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

Teaser

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 andneuronal excitability. Two key players act together in nanodomains, coupling Ca2+...

Summary

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

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).

Final results

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.

Website & more info

More info: http://www.ull.es/molcan.