Trifunctional Saxitoxin Conjugates for Covalent Labeling of Voltage‐Gated Sodium Channels**

Abstract

Voltage‐gated sodium ion channels (NaVs) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of NaVs hold potential value for understanding how channel regulation and post‐translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)‐saxitoxin (STX) – a potent and reversible inhibitor of multiple NaV isoforms – and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or ‘click’ functional group for labeling wild‐type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX‐maleimide‐coumarin derivative is most effective at irreversibly blocking Na+ conductance when applied to recombinant NaVs and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high‐affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next‐generation tool compounds for selective modification of NaVs expressed in the plasma membranes of cells.

Development of covalent chemogenetic K2Pchannel activators

“…Our development of the CATKLAMP covalent modification system provides a generalizable concept for the ion channel chemical biology toolbox 69 that should be applicable to any ion channel whose action is controlled by a small molecule ligand. We note that CATKLAMP may offer a means to label and track TREK subfamily channels by modifying the CAT335 scaffold with a second chemical handle using strategies similar to those used for saxitoxin-maleimide derivatives and voltage-gated sodium channels 70,71 . We also anticipate that leveraging the properties of this system to capture ligands, such as with tethering approaches 62-64 , should enable identification of new molecules to control K 2P function.…”