Optogenetic tools for manipulation of cyclic nucleotides, functionally
coupled to CNG-channels
Abstract
Background and Purpose The cyclic nucleotides cAMP and cGMP are
ubiquitous second messengers participating in the regulation of several
biological processes. Interference of cNMP signalling is linked to
multiple diseases and thus is an important component of pharmaceutical
research. The existing optogenetic toolbox in C. elegans is
restricted to soluble adenylyl cyclases, the membrane-bound
Blastocladiella CyclOp and hyperpolarizing rhodopsins, yet
missing are membrane-bound photoactivatable adenylyl cyclases and
hyperpolarizers on the basis of K+-currents.
Experimental Approach For the characterization of the
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 hyperpolarize the cells, we performed behavioural analyses
(locomotion, muscle contraction) and measured the cNMP content in vitro.
Key Results We implemented Catenaria CyclOp as a new
tool for cGMP production, allowing fine-control of cGMP levels. As
photoactivatable membrane-bound adenylyl cyclases, we established
YFP::BeCyclOp(A-2x) and YFP::CaCyclOp(A-2x), enabling more specific
optogenetic cAMP signalling compared to soluble ACs. For the
hyperpolarization of excitable cells by K+-currents,
we introduced the cAMP-gated K+-channel SthK from
Spirochaeta thermophila with either bPAC or BeCyclOp(A-2x), and
the Blastocladiella emersonii cGMP-gated
K+-channel BeCNG1 with BeCyclOp. Conclusion
and Implications We established a comprehensive suite of optogenetic
tools for cNMP manipulation for the nematode, which will be useful for
applications in many cell types, including sensory neurons which use
mainly cGMP as second messenger, and for potent hyperpolarization using
K+-ions.