Oxidation of multiple methionine residues impairs rapid sodium channel inactivation.
Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel Na(V)1.4 expressed in mammalian cells was studied by applying the Met-preferring oxidant chloramine-T or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of Met1470 in the putative receptor of the inactivation lid also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues in intracellular linkers connecting the membrane-spanning segments (442, 1139, 1154, 1316, 1469) were of minor importance. The results of mutagenesis, assays of other Na(V) channel isoforms (Na(V)1.2, Na(V)1.5, Na(V)1.7), and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of Na(V) channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.
Kassmann
Mario
M
Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena, Hans-Knöll-Str. 2, 07745, Jena, Germany.
Hansel
Alfred
A
Leipold
Enrico
E
Birkenbeil
Jan
J
Lu
Song-Qing
SQ
Hoshi
Toshinori
T
Heinemann
Stefan H
SH
D000465:Algorithms; D002478:Cells, Cultured; D002700:Chloramines / Q000494:pharmacology; D003627:Data Interpretation, Statistical; D004594:Electrophysiology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D016296:Mutagenesis; D062553:NAV1.4 Voltage-Gated Sodium Channel; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014105:Tosyl Compounds / Q000494:pharmacology
D002700:Chloramines; D005456:Fluorescent Dyes; D007546:Isoquinolines; D009124:Muscle Proteins; D062553:NAV1.4 Voltage-Gated Sodium Channel; C106251:SCN4A protein, human; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D014105:Tosyl Compounds; C016300:chloramine-T; C017475:lucifer yellow; D008715:Methionine