Going full circle - optogenetic control of Ca2+ release from and reuptake into the endoplasmic reticulum
1) Alexander Gottschalk (PI; University of Frankfurt), coworker: Frank Becker
2) Stefan Lehnart (PI; University of Göttingen), coworker: Miroslav Dura
3) Philipp Sasse (PI; University of Bonn), coworker: Wanchana Jangsangthong
In excitable cells, intracellular Ca2+ release and uptake from and into the endo/sarcoplasmic reticulum, via the Ryanodine Receptor (RyR) and SERCA-Ca2+-ATPase, respectively, are functionally coupled. This allows for tight spatiotemporal control of local Ca2+ signals (Ca2+ sparks) and safeguards cell viability through low resting Ca2+ concentrations. Despite the eminent role of RyR Ca2+ release channels and SERCA Ca2+-pumps in the brain, heart, pancreas and skeletal muscle, both in health and disease, their molecularly targeted manipulation remains challenging and complex. For example, patient mutations cause dangerous arrhythmias via RyR2 channels in the heart through increased Ca2+ leak. However, in experimental cellular models Ca2+ leak is commonly induced by non-physiological, typically pharmacological interventions at the cost of significant off-target effects. Importantly, the lack of RyR- and SERCA-specific tools compromises and delays the development of drug compounds for a large number of diseases, including arrhythmias, cancer, cognitive dysfunction, diabetes, epilepsy, heart failure, and muscle fatigue. While optogenetic manipulation of RyR channels and SERCA pumps addresses a challenging area, our collaborative efforts to develop the first OptoRyR tool are not only promising, but can open major opportunities for faster and combinatory strategies, with the vision to facilitate drug-safety testing.
In the first funding period we have developed de novo strategies for light-induced Ca2+ release. The most successful strategy used a Ca2+-conducting Channelrhodopsin2 variant in fusion to the RyR2. This OptoRyR2 relies on an endogenous amplification mechanism of Ca2+-induced Ca2+-release through cytosolic Ca2+-activation of RyR2 channels.
For the second funding period we will significantly extend the planned work through three major directions: In Aim 1 we will apply the mechanistic concept of OptoRyR2 to C. elegans and human stem cell-derived cardiomyocytes using CRISPR/Cas9 mediated gene editing to address fundamental biological questions in intact animals and to develop a novel optogenetic platform for drug-safety testing of RyR2-targeted chemical compounds. Aim 2 broadens the utility concept through a new mechanistic approach of direct opto-mechanical light-gating of RyR2 through insertion of LOV domains in the channel shell structure. This became possible through recent high-resolution CryoEM structures of RyR channels. Aim 3, literally closing the circle, extends the optogenetic toolbox to control Ca2+ uptake through two parallel strategies: light-activation of cAMP production molecularly linked to phospholamban and opto-mechanical control of SERCA will both modulate Ca2+ uptake. Thus, we will develop a toolbox for subcellular Ca2+ control and apply this in vitro and in vivo to enable exploring basic questions of Ca2+ homeostasis, as well as a new concept of drug-safety testing.
Logic of interactions:
OptoRyR and optoSERCA constructs will be generated for C. elegans and tested by the Gottschalk group, and in parallel, by the Sasse group, for the mouse. The Lehnart group will provide essential biophysical experiments (single-channel measurements) to characterize the mechanism of action of the generated constructs.
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