PKCε phosphorylation of the sodium channel NaV1.8 increases channel function and produces mechanical hyperalgesia in mice
Dai-Fei Wu, … , Sulayman D. Dib-Hajj, Robert O. Messing
Dai-Fei Wu, … , Sulayman D. Dib-Hajj, Robert O. Messing
Published March 19, 2012
Citation Information: J Clin Invest. 2012;122(4):1306-1315. https://doi.org/10.1172/JCI61934.
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Research Article Neuroscience

PKCε phosphorylation of the sodium channel NaV1.8 increases channel function and produces mechanical hyperalgesia in mice

Abstract

Mechanical hyperalgesia is a common and potentially disabling complication of many inflammatory and neuropathic conditions. Activation of the enzyme PKCε in primary afferent nociceptors is a major mechanism that underlies mechanical hyperalgesia, but the PKCε substrates involved downstream are not known. Here, we report that in a proteomic screen we identified the NaV1.8 sodium channel, which is selectively expressed in nociceptors, as a PKCε substrate. PKCε-mediated phosphorylation increased NaV1.8 currents, lowered the threshold voltage for activation, and produced a depolarizing shift in inactivation in wild-type — but not in PKCε-null — sensory neurons. PKCε phosphorylated NaV1.8 at S1452, and alanine substitution at this site blocked PKCε modulation of channel properties. Moreover, a specific PKCε activator peptide, ψεRACK, produced mechanical hyperalgesia in wild-type mice but not in Scn10a–/– mice, which lack NaV1.8 channels. These studies demonstrate that NaV1.8 is an important, direct substrate of PKCε that mediates PKCε-dependent mechanical hyperalgesia.

Authors

Dai-Fei Wu, Dave Chandra, Thomas McMahon, Dan Wang, Jahan Dadgar, Viktor N. Kharazia, Ying-Jian Liang, Stephen G. Waxman, Sulayman D. Dib-Hajj, Robert O. Messing

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Figure 4

Identification of PKCε phosphorylation sites in the L3 loop.

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Identification of PKCε phosphorylation sites in the L3 loop. (A) The top...
(A) The top 2 panels show an autoradiogram of phosphorylated intracellular L3 loop (p-L3) and a scanned image of a Coomassie blue–stained gel before autoradiography (L3). The bottom 2 panels are an autoradiogram and gel of PKCε phosphorylation of the large intracellular loop of the GABAA γ2 subunit (p-γ2s), a known PKCε substrate (9). (B) An autoradiogram and Western blot with anti-6xHis antibody, showing that PKCε phosphorylation of the NaV1.8 L3 loop was substantially reduced by alanine substitution at S1452 but not at T1437. (C) Quantification of L3 loop phosphorylation by PKCε. Results were calculated as the ratio of optical density values for phosphorylation (determined by autoradiography) and immunoreactivity (determined by Western blot analysis with anti-6xHis antibody) and were normalized to values obtained for native L3 loop run in parallel (mean ± SEM values from 3 experiments). *P = 0.0002 compared with a theoretical mean of 1.0 by 1-sample t test.