Cu 2+ (1,10 phenanthroline) 3 is an open‐channel blocker of the human skeletal muscle sodium channel

The formation of disulfide bridges is a classical approach used to study the mobility, proximity and distances of residues in a variety of proteins, including ligand‐ and voltage‐gated ion channels. We performed patch‐clamp studies to investigate the interaction of a pair of cysteines introduced into the human skeletal muscle voltage‐gated Na + channel (hNa v 1.4) using the oxidation catalyst, Cu 2+ (1,10‐phenanthroline) 3 (CuPhen). Our experiments resulted in a surprising finding, a reversible current inhibition of the mutant I1160C/L1482C containing two cysteines in the D3/and D4/S4–S5 loops, subjected to oxidative cross‐linking in the presence of CuPhen. We report here that CuPhen is an open channel blocker of both mutant and wild‐type (WT) hNa v 1.4 channels, however, for WT channels a more than 10‐fold higher concentration was needed to induce the same effect. Moreover, 1,10‐phenanthroline was capable of blocking Na + channels in the absence of Cu 2+ ions. Our results indicate a use‐ and voltage‐dependent binding and unbinding of CuPhen, reminiscent of the lidocaine quaternary derivative QX‐314 and the neurotoxin batrachotoxin. Care should be taken when using CuPhen as an oxidizing reagent in cross‐linking experiments, since it may directly affect channel activity. Our results identify CuPhen (and phenantroline) as a novel use‐dependent inhibitor of Na + channels, a mechanism that is shared by drugs widely used in the treatment of epilepsy, neuropathic pain, cardiac arrhythmia and myotonia. We hypothesize that I1160C in D3/S4–S5 and the corresponding L1482C mutation in D4/S4–S5 could allosterically affect a binding site located in the inner pore region of the channel.British Journal of Pharmacology (2006) 147 , 808–814. doi: 10.1038/sj.bjp.0706667