Non-technical summary The orchestrated activity of an ensemble of voltage-gated ion channels determines the initiation, shape and duration of the action potential in excitable cells. In primary pain-sensing neurons, this ensemble includes a high voltage-activated potassium channel. However, its molecular identity, function and modulation were unknown. Here, we show that the rapidly inactivating Kv3.4 channel underlying the high voltage-activated potassium current is a major determinant of action potential repolarization. Furthermore, we found that physiological activation of protein kinase C dramatically slows Kv3.4 channel inactivation, which enhances the channel's ability to influence action potential repolarization. Based on these results and earlier work, we conclude that phosphorylation of the Kv3.4 channel inactivation gate is a mechanism by which pain-sensing neurons shape action potential repolarization. This modulation will influence Ca2+-dependent processes that play vital roles in nociception and might become deregulated in chronic pain.
Abstract Fast inactivation of heterologously expressed Kv3.4 channels is dramatically slowed upon phosphorylation of the channel's N-terminal (N-type) inactivation gate by protein kinase C (PKC). However, the presence and physiological importance of this exquisite modulation in excitable tissues were unknown. Here, we employed minimally invasive cell-attached patch-clamping, single-cell qPCR and specific siRNAs to unambiguously demonstrate that fast-inactivating Kv3.4 channels underlie a robust high voltage-activated A-type K+ current (IAHV) in nociceptive dorsal root ganglion neurons from 7-day-old rats. We also show that PKC activation with phorbol 12,13-dibutyrate (PDBu) causes a 4-fold slowing of Kv3.4 channel inactivation and, consequently, accelerates the repolarization of the action potential (AP) by 22%, which shortens the AP duration by 14%. G-protein coupled receptor (GPCR) agonists eliminate IAHV fast inactivation in a membrane-delimited manner, suggesting a Kv3.4 channel signalling complex. Preincubation of the neurons with the PKC inhibitor bisindolylmaleimide II inhibits the effect of GPCR agonists and PDBu. Furthermore, activation of PKC via GPCR agonists recapitulates the effects of PDBu on the AP. Finally, transfection of the neurons with Kv3.4 siRNA prolongs the AP by 25% and abolishes the GPCR agonist-induced acceleration of the AP repolarization. These results show that Kv3.4 channels help shape the repolarization of the nociceptor AP, and that modulation of Kv3.4 channel N-type inactivation by PKC regulates AP repolarization and duration. We propose that the dramatic modulation of IAHV fast inactivation by PKC represents a novel mechanism of neural plasticity with potentially significant implications in the transition from acute to chronic pain.