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Optogenetic tools for manipulation of cyclic nucleotides, functionally coupled to CNG-channels
  • +7
  • Thilo Henss,
  • Jatin Nagpal,
  • Shiqiang Gao,
  • Ulrike Scheib,
  • Alessia Pieragnolo,
  • Alexander Hirschhäuser,
  • Franziska Schneider-Warme,
  • Peter Hegemann,
  • Georg Nagel,
  • Alexander Gottschalk
Thilo Henss
Goethe-Universitat Frankfurt am Main
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Jatin Nagpal
University College Cork
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Shiqiang Gao
University of Würzburg
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Ulrike Scheib
NUVISAN ICB GmbH
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Alessia Pieragnolo
Università degli Studi di Padova
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Alexander Hirschhäuser
Philipps-Universitat Marburg
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Franziska Schneider-Warme
University of Freiburg
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Peter Hegemann
Humboldt University of Berlin
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Georg Nagel
University of Würzburg
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Alexander Gottschalk
Goethe-Universitat Frankfurt am Main
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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.

Peer review status:ACCEPTED

06 Oct 2020Submitted to British Journal of Pharmacology
07 Oct 2020Submission Checks Completed
07 Oct 2020Assigned to Editor
08 Oct 2020Reviewer(s) Assigned
03 Nov 2020Review(s) Completed, Editorial Evaluation Pending
09 Nov 2020Editorial Decision: Revise Minor
10 Feb 20211st Revision Received
11 Feb 2021Submission Checks Completed
11 Feb 2021Assigned to Editor
11 Feb 2021Reviewer(s) Assigned
01 Mar 2021Review(s) Completed, Editorial Evaluation Pending
02 Mar 2021Editorial Decision: Accept