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

Since the advent of optogenetics, significant progress has been made in
developing tools to modulate and detect cellular activity using light. We
present a novel 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 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.