Abstract
Voltage-gated sodium ion channels (Na(V) s) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of Na(V) s 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 Na(V) 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 Na(V) s 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 Na(V) s expressed in the plasma membranes of cells.