Optogenetic tools for manipulation of cyclic nucleotides, functionally coupled to CNG-channels.

Abstract

BACKGROUND AND PURPOSE\nThe cyclic nucleotides cAMP and cGMP are ubiquitous second messengers that regulate numerous biological processes. Malfunctional cNMP signalling is linked to multiple diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in C. elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarising rhodopsins, yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarisers based on K+ -currents.\n\n\nEXPERIMENTAL APPROACH\nFor characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic-nucleotide gated channels in C. elegans muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to de- or hyperpolarise cells, we performed behavioural analyses (locomotion, muscle contraction), measured cNMP content in vitro, and compared in vivo expression levels.\n\n\nKEY RESULTS\nWe implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established photoactivatable membrane-bound adenylyl cyclases, based on mutated versions ( A-2x ) of Blastocladiella and Catenaria ( Be , Ca ) CyclOp, as N-terminal YFP fusion proteins, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarisation of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+ -channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the Blastocladiella emersonii cGMP-gated K+ -channel BeCNG1 together with BeCyclOp.\n\n\nCONCLUSION AND IMPLICATIONS\nWe established a comprehensive suite of optogenetic tools for cNMP manipulation, useful for applications in many cell types, including sensory neurons, and for potent hyperpolarisation.