Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

Abstract

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

White - cGMP Interaction Promotes Fast Locomotor Recovery from Anoxia in Adult Drosophila

“…Behavioral modulations by cGMP are well-documented. Upregulation of cGMP increases ion conductance and depolarizes membrane potential in Xenopus oocytes, and promotes muscle contraction in C. elegans [22]. High levels of cGMP activate cGMP-dependent protein kinase and promote locomotion while feeding in larvae and adult flies [23, 24].…”

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

“…The genomes of these microbes and its relative Catenaria anguillulae harbor genes that encode enzymerhodopsins consisting of a rhodopsin domain and a guanylylcyclase domain (without histidine kinase or response regulator domains) (71). In contrast to all other type 1 rhodopsins, the rhodopsin domains of all five known fungal enzymerhodopsins (three from A. macrogynus and one from each B. emersonii and C. anguillulae ) contain a predicted additional transmembrane helix in the N terminus (helix 0) (72). The cytoplasmic localization of the N terminal region has been confirmed by bimolecular fluorescence complementation, and its role in inhibition of the dark cyclase activity has been demonstrated by measurements from an N-terminally truncated version of the protein (72).…”

“…Be GC1 (under the names RhGC (74) and CyclOp (72)) produced the most robust elevation of the intracellular cGMP levels upon illumination of all tested homologous proteins, whereas its dark cyclase activity was very low, and no intrinsic ion channel or pumping activity was detected (72, 74). High specificity of its cyclase domain for cGMP over cAMP (cyclic adenosine monophosphate) was demonstrated by co-expression with two different subtypes of cyclic nucleotide-gated channel, each of which is specifically gated by one of the two cyclic nucleotides (74).…”

Molecular tools for acute spatiotemporal manipulation of signal transduction

“…Light-induced activation of GPCR signaling pathways, furthermore, is achieved through exogenous expression of naturally light-sensitive GPCRs, such as animal opsins, as well as chimeric GPCRs that combine the light sensitivity of an opsin to activate native G-proteins [44]. Lastly, exogenous overexpression of photoactivated adenylyl cyclases (PACs) [36] as well as guanylyl cyclases [45] allows for acute stimulation of cAMP and cGMP production, respectively, using light.…”