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Optoelectrical Dynamics of Ion channel Activation in Calcium Nanodomains

Periodic Reporting for period 4 - NANOPDICS (Optoelectrical Dynamics of Ion channel Activation in Calcium Nanodomains)

Reporting period: 2020-03-01 to 2021-08-31

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, during this project we have further determined 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. We have significantly advanced to optimize and deploy these novel optoelectrical reporters to study physiologically relevant Ca2+-induced processes both in cellular and animal models.
The NANOPDICS Project has succesfully consolidated the NANOPDICS-MOLCAN Lab, at the ITB and School of Medicine of the Universidad de La Laguna. The NANOPDICS team has been fully functional consisting of the PI, 5 postdocs, 2 PhD students, 1 technician and a Lab Manager. Main achievements include characterization of new sites involved in BK modulation by Calcium, the identification of independent movements triggered by specific activation of ion binding sites in the BK gating ring and/or voltage sensor (Miranda et al, PNAS 2016; Miranda et al, eLife 2018), super-resolution images of Cav-BK complexes in heterologous expression systems (Kshatri et al, 2018, JGP; another manuscript is currently ready for submission), characterization of Cav-BK complexes composed of various Cav types and NMDAR-BK complexes (Kshatri et al 2018, JGP; Gomez et al, 2021, PNAS; Kshatri et al, manuscript in preparation), unnatural aminoacids insertion into BK channels and successful functional assays (Gonzalez-Hernandez et al, Doctoral Thesis defended September 2021; manuscript in preparation), development of potential new BK-Ca sensors based on BK channel proteins (Roger Gimeno’s PhD Thesis defended 2019). In addition, we have started the generation of a transgenic mouse with fluorescently labelled endogenous BK channels (B. Rivero, undergoing work in the laboratory). Finally, and as one of the most relevant contributions of our lab, we have presented the first functional evidence for a role of BK channels in synaptic plasticity, which has open a new avenue of research in the laboratory (Gomez et al, 2021, PNAS).
The NANOPDICS lab and project has been presented as invited seminars at many international academic institutions. Conversely, several renowned researchers have visited the facilities and imparted specialized seminars for the project. The project has brought prestige to our lab and institution, and has been very well recognised within the ion channels, Biophysics and Neuroscience research communities, resulting in very relevant professional achievements of the PI and lab members. In addition to prestigious scientific publications, NANOPDICS has extensively participated in many other activites to disseminate our results, including participation in large number of scientific conferences, workshops and courses; strong presence in the media via interviews, press releases and other activities; participacion in science outreach in our community, such as TEDex talks, Pint of Science editions, series of talks at schools and high-schools, etc. Our activity and public engagement has resulted in three relevant recognitions to the PI and other members of the research group.
At the moment this summary is elaborated, the project has achieved a unique lab setup to perform electrophysiological recordings from oocytes, cell lines, neuron cultures and brain slices preparations. These recordings can be performed simultaneously with fluorescence approaches, including super-resolution (STORM). We are generating novel transgenic mouse lines. In its current state, the lab facilities provide a service to the scientific community at large. the team has worked very well and in perfect coordination and collaboration, which has constituted a significant advantage towards good progress. The post docs have published results, produced more work that will be submitted soon, and at the same time are accomplishing well-timed, specialized training of the PhD students; the technicians have been very efficient in managing the lab facilities and ensuring proper functioning of the equipment and availability of supplies. Two PhD students have defended their PhD Thesis during this action, and a third has indirectly benefited by the scientific and competitive atmosphere created by the NANOPDICS actions. A total of 18 articles and one book chapter with involvement or influence of the NANOPDICS team have been published in this period. One manuscript is currently under review, plus another four manuscripts are being prepared for submission. The action has had significant international impact through a series of invited talks by the PI at international meetings, seminars at highly-recognized institutions, workshops and specialized courses. The PI has achieved a significant level of recognition by peers, institutions and scientific societies, establishing a highly influential position. The PI has secured a tenured professorship at the HI during the course of the action. The project website has provided further international impact.
Kenton Swartz visit
NANOPDICS PI in Pint of Science 2018
Ricardo Gomez in Chile
NANOPDICS contacts
NANOPDICS technology
ACIISI director visit
Alejandro Cerrada in SBE Congress
Tim Marzullo's visit
PI Teresa Giraldez
PI cited within the 30 most influential women in the history of the Canary Islands
Lluis Montoliu visit