Sulfhydryl modulation of K⁺ channels in rat ventricular myocytes

Oxidative stress markedly alters protein function through redox modification of sulfhydryl groups present in cysteine residues. To explore the role of redox state in modulating cardiac K⁺ channels, this study examined the effects of sulfhydryl modifiers on the repolarizing transient outward current (I_to) in voltage-clamped myocytes from rat ventricle. Oxidized glutathione (GSSG; 5 mM), an endogenous disulfide that specifically reacts with protein sulfhydryls, decreased maximum I_to amplitude from baseline by 49% when added to the external solution (P < 0.05) and by 27% during internal dialysis (P < 0.05). The membrane-impermeable disulfide, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) did not alter I_to when added to the external solution, but it decreased current amplitude by 31% during internal dialysis (P < 0.05). GSSG-mediated I_to inhibition varied in a frequency- and voltage-dependent manner, consistent with a state-dependent blocking mechanism. This phenomenon was also observed in myocytes internally dialyzed with DTNB or Cd²⁺, which also covalently binds to free sulfhydryls. Inhibition of I^to by GSSG was not reversed by washout alone, consistent with the stable nature of covalently-modified sulfhydryl groups. However, when myocytes pretreated with GSSG were dialyzed with the reducing agent dithiothreitol, I_to amplitude increased significantly by 42% (P < 0.05). These data suggest that α-subunits underlying I_to, or associated proteins, have one or more sulfhydryl groups within the cytoplasmic domain that directly modulate channel activity in response to changes in cell redox state. Redox modulation of I_to channels may be an important post-translational mechanism contributing to acute changes in cardiac repolarization under conditions of oxidative stress, such as ischemia and reperfusion.