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This is the final published version of the article from the Journal of Neuroscience, 2022 Oct 3;42(46):8647-8657.

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The Kv3.4 channel regulates action potential (AP) repolarization in nociceptors and excitatory synaptic transmission in the spinal cord. We hypothesize that this is a tunable role governed by protein kinase-C-dependent phosphorylation of the Kv3.4 cytoplasmic N-terminal inactivation domain (NTID) at four nonequivalent sites. However, there is a paucity of causation evidence linking the phosphorylation status of Kv3.4 to the properties of the AP. To establish this link, we used adeno-associated viral vectors to specifically manipulate the expression and the effective phosphorylation status of Kv3.4 in cultured dorsal root ganglion (DRG) neurons from mixed-sex rat embryos at embryonic day 18. These vectors encoded GFP (background control), wild-type (WT) Kv3.4, phosphonull (PN) Kv3.4 mutant (PN = S[8,9,15,21]A), phosphomimic (PM) Kv3.4 mutant (PM = S[8,9,15,21]D), and a Kv3.4 nonconducting dominant-negative (DN) pore mutant (DN = W429F). Following viral infection of the DRG neurons, we evaluated transduction efficiency and Kv3.4 expression and function via fluorescence microscopy and patch clamping. All functional Kv3.4 constructs induced current overexpression with similar voltage dependence of activation. However, whereas Kv3.4-WT and Kv3.4-PN induced fast transient currents, the Kv3.4-PM induced currents exhibiting impaired inactivation. In contrast, the Kv3.4-DN abolished the endogenous Kv3.4 current. Consequently, Kv3.4-DN and Kv3.4-PM produced APs with the longest and shortest durations, respectively, whereas Kv3.4-WT and Kv3.4-PN produced intermediate results. Moreover, the AP widths and maximum rates of AP repolarization from these groups are negatively correlated. We conclude that the expression and effective phosphorylation status of the Kv3.4 NTID confer a tunable mechanism of AP repolarization, which may provide exquisite regulation of pain signaling in DRG neurons.

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