title: Cu2+ (1,10 phenanthroline)3 is an open-channel blocker of the human skeletal muscle sodium channel.
abstract: 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, Cu2+ (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 Cu2+ 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.
authors.affiliation: Department of Neurology and Applied Physiology, University of Ulm, D-89069 Ulm, Germany.
authors.forename: Mariana Oana
authors.initials: MO
authors.lastname: Popa
authors.forename: Holger
authors.initials: H
authors.lastname: Lerche
mesh_terms: D000818:Animals; D003300:Copper / Q000494:pharmacology; D003627:Data Interpretation, Statistical; D004594:Electrophysiology; D006801:Humans; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D009124:Muscle Proteins / Q000187:drug effects / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects*; D016297:Mutagenesis, Site-Directed; D062553:NAV1.4 Voltage-Gated Sodium Channel; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010618:Phenanthrolines / Q000494:pharmacology*; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D014981:Xenopus
chemical_list: D009124:Muscle Proteins; D062553:NAV1.4 Voltage-Gated Sodium Channel; D010618:Phenanthrolines; C106251:SCN4A protein, human; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C038556:bis(1,10-phenanthroline)copper(1+) ion; D003300:Copper