Anna Moroni (Department of Biosciences, University of Milan, Italy) has been invited by Filippo Del Bene to hold a talk on Monday 17th April, 11.00 AM, in the conference room of the UCL, 13 Rue Moreau.

This talk will be on "Engineering K+ channels for long term inhibition of cell excitability".

Abstract

Opening of potassium (K+) channels universally reduces cell excitability leading to long lasting inhibition of neuronal activity. Remote activation of K+ channels represents a useful research tool with great potentiality in clinics. Our aim is to develop K+ channel proteins that can be regulated in remote by external stimuli. We use different strategies based on de-novo protein engineering of channels, structure-inspired modulation of native channels and regulation of transcription. By one approach we add exogenous protein domains to the pore of the small KCV channel and we evolve the chimeric protein by means of protein engineering techniques. We have obtained channels regulated by light, temperature and ROS. We have optimized this system in such a way that it expresses in neurons and allows long lasting light-off activity and, in turn, long lasting inhibition of neurons. A second approach is based on light-dependent modulation of native HCN channels. These channels that control pacemaking activity in brain and heart, have a regulatory subunit TRIP8b that prevents cAMP induced enhancement of their current. We have reduced TRIP8b to a short peptide, nanoTRIP, and fused it to a LOV2 domain that releases the active peptide upon blue light. A final strategy uses a light-dependent dimerization system to activate the transcription of small K+ channels. With this system we can target active K+ channels not only to the plasma membrane but also to the inner mitochondrial membrane. This approach allows long lasting depolarization of the mitochondria with physiological consequences, such as modulation of mitochondrial calcium content.

Personal Statement

My laboratory works on structure-function of potassium (K+) channels, a class of ubiquitous membrane proteins that control basic cellular functions, in particular membrane excitability in neurons and myocytes. We identified and characterized K+ channel from viruses (Plugge et al., Science, 2000), plants (Saponaro et al, Plant Cell, 2017) and animals (Saponaro et al, 2021 Molecular Cell). In our hands, viral K+ channels became an established model system for more complex mammalian channels. More recently, we employed viral channels as a protein module to engineer several synthetic channels including BLINK, a light-gated K+ channel for optogenetics (Cosentino et al., Science 2015, Alberio et al, 2018 Nature Methods). We are presently engineering synthetic channels activated by external stimuli, ultrasounds and magnetic fields. The second line of research concerns the HCN channels that control pacemaker activity in heart and brain. By combining functional recordings (patch clamp) to structural studies (X-ray crystallography and single particle cryoEM), we unraveled the mechanism of cAMP regulation and identified small molecules, peptide drugs and nanobodies that revert the effect of pathological mutations (Lolicato et al., Nat Chem Biol, 2014; Saponaro et al, PNAS 2014; Porro et al, Elife 2019). More recently we solved the structure of HCN4, the sinoatrial node subtype, with the pore in the closed and in the open state (Saponaro et al, Molecular Cell 2021) and in complex with the marketed drug ivabradine (unpublished).