Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

Abstract

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.

Modulating protein activity and cellular function by methionine residue oxidation

“…But after channel protein oxidation by Ch-T, a dose-dependent slowing down of the inactivation process has been observed for Na v 1.4. Similar oxidant effects were also observed in Na v 1.2, Na v 1.5_C373Y, and Na v 1.7 (Kassmann et al 2008). Such oxidant effect on Na v 1.4 was progressive with time and irreversible even after DTT treatment, indicating non-involvement of Cys residues.…”

“…Simultaneous mutation of Met1305L, Met1469L, and Met1470L in Na v 1.4 relieved the oxidant effect. Lucifer yellow-mediated photodynamic action had a similar effect in Na v 1.4 as Ch-T (Kassmann et al 2008).…”

Human voltage-gated sodium channel mutations that cause inherited neuronal and muscle channelopathies increase resurgent sodium currents

“…In addition, our data - in conjunction with the previous study indicating that β-pompilidotoxin can artificially induce resurgent currents in cerebellar Purkinje neurons (9) - indicate that any manipulation that slows or destabilizes inactivation has the potential to induce resurgent currents. Many posttranslational modifications have been reported to slow the rate of inactivation or increase the amplitude of persistent sodium currents, including hypoxia (33), phosphorylation (34), altered calcium signaling (35), G protein activation (36), and oxidation (37). We propose that these alterations could also result in abnormal resurgent current generation.…”

Abnormal Ca2+Cycling in Failing Ventricular Myocytes: Role of NOS1-Mediated Nitroso-Redox Balance

“…These proteins are also regulated by ROS. The voltage-gated Na + channel (Nav1.5) inactivation is greatly impeded by oxidation of methionine residues (increasing Na + entry) (56,88 While ROS can directly modulate ECC protein function via oxidation (i.e., redox signaling), it is also known that various kinases and phosphatases are redox sensitive. Thus, there may be an additional layer of complexity of ROS in which it may also regulate ECC protein function via changing phosphorylation status.…”