title: Towards the engineering of a magneto sensitive potassium channel
Creators: name: Brocca, Lorenzo
Publisher: name: UNSPECIFIED
Year: 2022

description: Optogenetics is the most widely used technique employed by neuroscientists to control neuronal activity with high spatial and temporal resolution either in vitro and in vivo. Optogenetics relies on naturally occurring or synthetic ion channels whose gating is controlled by light. Both excitatory and inhibitory optogenetic tools are currently available to remotely promote or prevent neuronal firing. Inhibitory tools are of particular interest as therapeutic agent for the treatment of neuropathic pain, a pathology caused by nerve lesion/inflammation that generates uncontrolled firing in peripheral sensory neurons. Given the limited penetration of light into tissues that greatly hampers the usage of optogenetic tools in vivo, the search is on for novel, alternative ways to stimulate inhibitory tools. Magneto-genetics was recently proposed as an alternative technique to optogenetics. It relies on the capability of external magnetic fields, which deeply penetrate into tissues, to activate ion channels. Initially, the heat-activated channel TRPV1 was conjugated to exogenously supplied iron oxide nanoparticles. The latter generate heat in response to an oscillating magnetic field and this rise in temperature is responsible for the opening of TRPV1. In the second generation of magneto-genetic tools the TRPV channels (TRPV1-4) have been linked to ferritin, a 24-mer protein forming an hollow sphere that can store up to 4500 iron atoms. Activation of ferritin-engineered TRPV channels by oscillating magnetic fields was reported to occur both in vitro and in vivo experiments. The physical principle behind the working mechanism of magneto-genetic tools is unclear and presently debated. However, in the case of Magneto2.0, a TRPV4-based channel, it was shown experimentally that opening is due to the rise in temperature, generated by ferritin upon exposure to an oscillating magnetic field. To date, all magneto-genetic tools use TRPV channels, which mainly conduct calcium, magnesium and sodium ions, limiting the spectrum of application of these tools to neuronal excitation. The aim of this work is to generate a potassium (K⁺) magneto-genetic tool, with the long term aim to use it in the treatment of neuropathic pain. In nature, all known temperature-sensitive potassium channels are polymodal, i.e., they respond to a number of stimuli, beside temperature. This finding motivated us to engineer a member of the viral potassium channel family, Kcv Next to Smith (KcvNTS), into a temperature sensitive channel. To this end, we fused at its C terminus the cytosolic domain of the temperature sensitive bacterial sodium channel NavAe1. One of the engineered constructs, called v1, showed the wanted property of being closed at 37°C and open &gt;45°C. We introduced single point mutations in the C-terminal of v1 to fine-tune the temperature range of gating. Mutation M267A decreased the opening temperature of the channel from 45°C to 40°C, making this a good tool for mammals. The M267A mutant was renamed “Temperature Induced Channel for K⁺” (TICK1). TICK1 activation and deactivation kinetics were characterized, showing an activation τ of around 10 seconds and a variable deactivation τ, ranging from 66 to 672 seconds. In parallel, we also characterized the ferritin that we intended to connect to TICK. According to Stanley et al, 2015, the moiety is formed by a dimer, obtained by the fusion of light and heavy chain monomers. This ensures a light/heavy chain ratio of 1:1, which was reported to increase the iron uptake. We further added a EGFP at the N terminus of the dimer and demonstrated that this construct, construct EGFP-mFT, assembles into 12mer ferritins in HEK293 cells. We then tested by correlation spectroscopy if magnetic fields affect the engineered ferritins, but no changes in diffusion coefficients were found in the presence of static magnetic fields. Purified ferritins were further analyzed by Inductively coupled plasma - optical emission spectrometry (ICP-OES) to measure their iron content, revealing that each ferritin in average contains 583 atoms of iron, a result in accordance with the physiological iron content of mammalian ferritins. As a next step we built our magneto-genetic tool: TICK1 was modified with an N-terminal EGFP nanobody (nanoTICK1) and this construct was co-expressed in HEK293T cells with EGFP-mFT. The nanobody ensures the binding of ferritin to the channel that was renamed MagKCV. After preliminary testing proper MagKCV membrane localization and temperature activation, we investigated the magnetic properties of this synthetic channel. An experimental setup was built with a cylindrical permanent magnet manually moved back and forth from the cell at 1Hz generating a maximum magnetic field of around 50mT. Experiments were conducted with cells maintained at 37°C. Around 20% of tested cells expressing MagKCV showed an activation of the channel in response to magnetic field. The activation of MagKCV is slower than the one of TICK1, but still maintains the typical TICK1 current-voltage relationship and barium sensitivity. In the control condition, with no magnetic field exposure, 1cell over 40 tested (2,5%) showed an open channel, indicating that MagKCV might be slightly less regulated than TICK1 (100% of temperature regulation, n=37). MagKCV appears to be a promising prototype for the development of the first K⁺ magneto-genetic tool.