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Optogenetic silencing by combining a rhodopsin cyclase with an engineered cGMP-gated potassium channel.

Science advances

Authors: Anika Spreen, Nidish Ponath Sadanandan, Martin Winfried Schneider, Enrico Kuehn, Andries Napo Leemisa, Roberta De Zio, Niklas Meyer, Wayne Busse, Bela Erlinghagen, Lea Adenauer, Thoralf Opitz, Wolfgang Bönigk, Enrico Schiewer, Jonas Heer, Dietmar Schmitz, Johannes Vierock, Heinz Beck, Peter Hegemann, Herwig Baier, Franziska Schneider-Warme, Yinth Andrea Bernal Sierra, Reinhard Seifert

Since the advent of optogenetics, great progress has been made in developing tools to modulate and detect cellular activity using light. We present a two-component optogenetic silencing tool, RoCK (rhodopsin cyclase/K channel), which pairs the rhodopsin-guanylyl cyclase CaRhGC with customized SthK K channels that are engineered to open selectively upon guanosine 3',5'-monophosphate (cGMP) binding. By enhancing the cGMP sensitivity and open probability of SthK mutants, we obtained four channel variants suited for different levels of cGMP concentration. CaRhGC's membrane-bound nature enables localized cGMP production, and the lack of dark activity reduces the risk for off-target effects. Optimized RoCK effectively modulated cellular activity in mouse hippocampal neurons, in acute hippocampal slices, and in rabbit cardiomyocytes. In zebrafish, RoCK silenced motor neurons in vivo, suppressing the characteristic coiling behavior of embryos, thus highlighting its potential for behavioral studies. In summary, RoCK expands our optogenetic toolkit threefold for fast cGMP production, fast cGMP sensing, and K-based cell silencing.

PMID: 41313760

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