A Triple Cysteine Motif As Major Determinant of the Modulation of Neuronal K7 Channels by the Paracetamol Metabolite N-acetyl-p-benzo Quinone Imine

Background And Purpose: The analgesic action of paracetamol involves K7 channels, and its metabolite N-acetyl-p-benzo quinone imine (NAPQI), a cysteine modifying reagent, was shown to increase currents through such channels in nociceptors. Modification of cysteine residues by N-ethylmaleimide, HO, or nitric oxide has been found to modulate currents through K7 channels. The study aims to identify whether, and if so which, cysteine residues in neuronal K7 channels might be responsible for the effects of NAPQI.

Experimental Approach: To address this question, we used a combination of perforated patch-clamp recordings, site-directed mutagenesis, and mass spectrometry applied to recombinant K7.1 to K7.5 channels.

Key Results: Currents through the cardiac subtype K7.1 were reduced by NAPQI. Currents through all other subtypes were increased, either by an isolated shift of the channel voltage dependence to more negative values (K7.3) or by such a shift combined with increased maximal current levels (K7.2, K7.4, K7.5). A stretch of three cysteine residues in the S2-S3 linker region of K7.2 was necessary and sufficient to mediate these effects.

Conclusion And Implication: The paracetamol metabolite N-acetyl-p-benzo quinone imine (NAPQI) modifies cysteine residues of K7 subunits and reinforces channel gating in homomeric and heteromeric K7.2 to K7.5, but not in K7.1 channels. In K7.2, a triple cysteine motif located within the S2-S3 linker region mediates this reinforcement that can be expected to reduce the excitability of nociceptors and to mediate antinociceptive actions of paracetamol.