Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKM zeta

Zhang YH, Kays J, Hodgdon KE, Sacktor TC, Nicol GD. Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKM zeta. J Neurophysiol 107: 315-335, 2012. First published October 5, 2011; doi:10.1152jn. 00030.2011.-Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK12 (PD-98059), PLC (U-73122, neomycin), or conventionalnovel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKM zeta, PKC zeta, and PKC lambda/iota) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKC zeta. Both Western blots and quantitative real-time PCR established that PKM zeta, but neither full-length PKC zeta nor PKC lambda/iota, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKC zeta or PKC iota augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKC zeta suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKC zeta. These results demonstrate that NGF leads to the activation of PKM zeta that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.