Cysteine-modification of Kv7 Channels as Analgesic Mechanism of Action of Acetaminophen

Abstract

N-acetyl-p-benzoquinone imine (NAPQI), an active metabolite of acetaminophen, decreases excitability in first- and second-order neurons of the nociceptive pathway, which involves an activation of Kv7 channels. NAPQI modifies cysteine residues and both alkylation and oxidative modification of cysteine residues in Kv7 channels were shown to increase M-currents. The aim of the study is to determine, if modification of cysteine residues in Kv7 channels by NAPQI is responsible for the reported increase of Kv7 currents. Heterologously expressed Kv7.1 to Kv7.5 channels were recorded in the perforated voltage clamp mode and clamped to −30 mV. Once every 15 s, the cells were hyperpolarized to −80 mV to elicit a deactivation current. Activation curves were recorded between −150 mV and +50 mV in 10 mV increments. Current levels at +60 mV were evaluated. Activation curves were recorded before, after three and ten minutes of NAPQI application. The effects of NAPQI were tested on wt and mutated versions of the respective Kv7 channel subtypes. While currents through Kv7.3 channels remained unchanged and Kv7.1-currents were reduced, deactivation currents through heterologously expressed Kv7.2, Kv7.4 and Kv7.5 channels increased about two-fold after ten minutes of NAPQI application. All currents remained unchanged during a subsequent five-minute wash out phase. Maximal current levels of Kv7.2, 7.4, and Kv7.5 homomeric channels were increased but only Kv7.2 and Kv7.4 also showed a left shift in half-maximal channel activation. A triple-cysteine stretch in the S2-S3 linker region was identified as responsible for the NAPQI-mediated increase of Kv7 currents.This module of three subsequent cysteine residues was previously shown to mediate the current increase induced by oxidative modification. We conclude that covalent modification of this stretch of three cysteine residues is responsible for the increase of Kv7 currents in response to NAPQI application.